Conformation specific antibodies

ABSTRACT

The disclosure provides, inter alia, binding proteins (e.g., antibodies) that bind to an integrin in an activated conformation, e.g., activated LFA-1 (“aLFA-1 ”), e.g., relative to a non-activated conformation of LFA-1. In one embodiment, the binding proteins inhibit at least one function of an aLFA-1, e.g., inhibit a binding interaction between aLFA-1 and a cognate ligand of aLFA-1, e.g., an ICAM protein. The binding proteins can be used to treat or prevent an inflammatory disorder or other disorder.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/589,956, now U.S. Pat. No. 7,927,591, filed Sep. 20, 2007, which is a371 of International Application No. PCT/US2005/05361, filed Feb. 22,2005, which claims priority to U.S. application Ser. No. 60/546,354,filed Feb. 19, 2004, the contents of which are hereby incorporatedherein in their entirety by reference.

BACKGROUND

Integrins are cell surface molecules that mediate important interactionsbetween cells and between cells and the extracellular milieu. Integrinscan adopt at least two different conformations on cell surfaces: anon-activated conformation that does not bind to the integrin ligand andan activated conformation that can bind the integrin ligand. Cellularsignalling can cause integrins to alter their conformation from anon-activated conformation to an activated conformation. Afteractivation, integrins bind in a specific manner to their cognate ligandson the surface of other cells, in the extracellular matrix, or that areassembled in the clotting or complement cascades.

Each integrin includes an subunit and a β subunit. Over twenty differentintegrin heterodimers are known. Many integrins are selectivelyexpressed on particular cells in the body. For example, a subset ofintegrins are selectively expressed on leukocytes.

Integrins on leukocytes are of central importance in leukocyteemigration and in inflammatory and immune responses. Two exemplaryintegrins on leukocytes are LFA-1 and Mac-1. LFA-1 (αLβ2) binds to anumber of cognate ligands, including inflammation-associated cellsurface molecules (ICAM), e.g., ICAM-1, ICAM-2, ICAM-3, ICAM-4, andICAM-5. Mac-1 (αMβ2) binds ICAM-1, the complement component iC3b, andthe clotting component fibrinogen.

SUMMARY

Disclosed are binding proteins that interact with integrins (“integrinbinding proteins”), particularly specific conformations of integrins. Anexemplary integrin binding protein is an antibody. An integrin bindingprotein can preferentially interact with an activated conformation of anintegrin, e.g., relative to a non-activated conformation, e.g., aninactive or resting conformation. An integrin binding protein canpreferentially interact with a mimic of an activated conformation of anintegrin (e.g., a modified integrin whose conformation is constrained ina state competent to bind to a cognate ligand), e.g., relative to anon-activated conformation, e.g., inactive or resting conformation, ormimics thereof. The integrin binding protein can bind with at least 1.5,2, 3, 4, 5, 10, 15, 20, 50, 70, 80, 100, 500, 1000, or 10⁵ fold greateraffinity to the preferred conformation relative to the disfavoredconformation.

In one embodiment, the integrin binding protein can be used to modulateintegrin activity, e.g., antagonize an activity of an activatedintegrin. For example, the integrin binding protein can be used toinhibit interaction between a cell that has an activated integrin on itssurface and a cognate ligand of the activated integrin.

In one embodiment, the integrin binding protein interacts with aleukocyte integrin, e.g., LFA-1, e.g., activated LFA-1 (“aLFA-1”), e.g.,human aLFA-1.

In one embodiment, the integrin binding protein is an antibody. Theantibody can include one or more human regions, e.g., one or more humanCDRs, one or more human frameworks (e.g., germline or somaticallymutated human FR), or one or more human constant regions, or effectivelyhuman regions of the same.

In one embodiment, the integrin binding protein inhibits aLFA-1activity. For example, the integrin binding protein prevents aLFA-1 frominteracting with a binding partner, e.g., a cognate ligand of LFA-1. Inparticular cases, the antibody can prevent aLFA-1 from interacting withan ICAM, e.g., ICAM-1, ICAM-2, ICAM-3, ICAM-4, or ICAM-5.

The integrin binding protein can modulate (e.g., decrease) inflammation,and accordingly can be used to treat an inflammatory disorder, e.g.,rheumatoid arthritis or psoriasis. Accordingly, the integrin bindingprotein can be administered to a subject in an amount effective to treator prevent such a disorder.

In one aspect, the disclosure features a protein that includes animmunoglobulin heavy chain (HC) variable domain sequence and animmunoglobulin light chain (LC) variable domain sequence. The HCvariable domain sequence and the LC variable domain sequence form anantigen binding site that binds to an activated conformation of LFA-1(“aLFA-1”), e.g., with cation dependence (e.g., that detectably binds at10 μg/ml protein concentration). For example, maximal binding requiresthe presence of a cation. The protein can require magnesium or manganesefor binding to LFA-1. Exemplary cation concentrations is between 0.01and 11 mM, e.g., between 0.1 and 5 mM, or 0.1 and 3 mM. In oneembodiment, the protein binds to LFA-1 in the presence of magnesium,EGTA and the CBRLFA-1/2 antibody, but not in the presence of magnesium,calcium, and the CBRLFA-1/2 antibody.

In one embodiment, the proteins binds to aLFA-1 with a better affinitythan MHM24. For example, the protein binds with a K_(D) that is lessthan the K_(D) of MHM24, e.g., at least 0.1, 0.5, or 1 nM less than theK_(D) of MHM24.

In one embodiment, the protein can bind to a K287C/K294C I-domain of αL.For example, the protein preferentially binds a K287C/K294C I-domain ofαL relative to L161C/F299C I-domain of αL or wild-type αL.

In one embodiment, the protein binds aLFA-1 with a K_(D) of less than10⁻⁷, 10⁻⁸, 10⁻⁹, 10⁻¹⁰, 10⁻¹¹, or 10⁻¹² M. In some cases, the proteinbinds human aLFA-1 with a k_(off) of less than 10, 5, 1, 0.5, 0.2, 0.1,or 0.05 s⁻¹. In one embodiment, the protein can reduce interactionbetween LFA-1 and a cognate ligand of LFA-1 (e.g., an ICAM, e.g.,ICAM-1). In one embodiment, the protein can reduce interaction between aleukocyte and an ICAM-expressing cell, e.g., an endothelial cell.

In one embodiment, the H1 and H2 hypervariable loops of the HC variabledomain sequence have the same canonical structure as an antibodydescribed herein. For example, the heavy chain variable domain sequencefowls a variable domain having the 1-3 Chothia canonical structure forthe H1 and H2 hypervariable loops.

In one embodiment, the L1 and L2 hypervariable loops of the LC variabledomain sequence have the same canonical structure as an antibodydescribed herein. For example, the light chain variable domain sequenceforms a variable domain having the 2-1 Chothia canonical structure forthe L1 and L2 hypervariable loops.

In another aspect, the disclosure features a protein that includes animmunoglobulin heavy chain (HC) variable domain sequence and animmunoglobulin light chain (LC) variable domain sequence, wherein the HCvariable domain sequence and the LC variable domain sequence form anantigen binding site that binds to an activated conformation of LFA-1,wherein the heavy chain variable domain sequence includes (a) a CDR1that includes at least 3, 4, or 5 amino acids (of 5) from RYVMW (SEQ IDNO:1), (b) a CDR2 that includes at least 13, 14, 15, 16, or 17 aminoacid (of 17) from YIWPSGGNTYYADSVKG (SEQ ID NO:2), and/or (c) a CDR3that includes at least 5, 6, 7, 8, 9, 10, 11 amino acids (of 11) fromSYDFWSNAFDI (SEQ ID NO:3) or another CDR3 described herein (e.g., froman affinity matured variant of D2-57). The protein can include otherfeatures described herein.

Exemplary sequences in the region of CDR3 of the heavy chain variabledomain sequence can include Xa-S-X2-D-X4-X5-S-X7-A-X8-X9-X10-X11 (SEQ IDNO:4). X can be any amino acid, preferably any non-cysteine amino acid.The sequence can have one or more of the following properties:

(i) Xa is hydrophilic, e.g., an uncharged hydrophilic residues such as Sor N;

(ii) X2 is aromatic, e.g., Y or F;

(iii) X4 is hydrophobic (e.g., L or aromatic, e.g., Y or F);

(iv) X5 is hydrophobic, e.g., a large hydrophobic side chain, e.g., W orR;

(v) X7 is N or Y, or another amino with a side chain that includes ahydroxyl;

(vi) X9 is aromatic, e.g., Y or F;

(vii) X10 is a small residue, e.g., a polar residue such as D or E, orA; and

(viii) X11 is any amino acid, e.g., K, I, S, M, N, V, or L.

The sequence can includeS-(Y/F)-D-(L/Y/F)-(W/R/K)-S-(N/Q/Y)-A-(Y/F)-(D/E/A)-(K/I/S/M/N/V/L) (SEQID NO:5) orS-(Y/F)-D-(L/Y/F)-(W/R)-S-(N/Y)-A-(Y/F)-(D/E/A)-(K/I/S/M/N/V/L) (SEQ IDNO:6). Still another sequence can include:Xa-(S/T)-X2-(D/E)-X4-X5-(S/T)-X7-(G/A/S)-X8-X9-X10-X11.

In one embodiment, the protein includes features of D2-57 or DX-2001,e.g., the CDR regions of the D2-57 antibody. In one embodiment, theheavy and light chain variable domain sequences are at least 70, 80, 85,90, 92, 93, 94, 95, 97, 98, 99, or 100% identical to correspondingvariable domain sequences of the D2-57 or the DX-2001 antibody.

In another aspect, the disclosure features a protein including animmunoglobulin heavy chain (HC) variable domain sequence and animmunoglobulin light chain (LC) variable domain sequence, wherein the HCvariable domain sequence and the LC variable domain sequence form anantigen binding site that binds to an activated conformation of LFA-1,wherein the light chain variable domain sequence includes (a) a CDR1that includes at least 7, 8, 9, 10, or 11 amino acids (of 11) fromRASQSIGSYLN (SEQ ID NO:7), (b) a CDR2 that includes at least 4, 5, 6, or7 amino acids (of 7) from AASSLQS (SEQ ID NO:8), and/or (c) a CDR3 thatincludes at least 5, 6, 7, or 8 (of 8) amino acids from QQSYSTPS (SEQ IDNO:9). The protein can include other features described herein. In oneembodiment, the protein includes features of D2-57 or DX-2001, e.g., theCDR regions of the D2-57 or the DX-2001 antibody. In one embodiment, theheavy and light chain variable domain sequences are at least 70, 80, 85,90, 92, 93, 94, 95, 97, 98, 99, or 100% identical to correspondingvariable domain sequences of the D2-57 or the DX-2001 antibody.

In another aspect, the disclosure features a protein that includes animmunoglobulin heavy chain (HC) variable domain sequence and animmunoglobulin light chain (LC) variable domain sequence, wherein the HCvariable domain sequence and the LC variable domain sequence form anantigen binding site that binds to an activated conformation of LFA-1,wherein the heavy chain variable domain sequence includes (a) a CDR1that includes at least 3, 4, or 5 amino acids (of 5) from HYGMS (SEQ IDNO:10), (b) a CDR2 that includes at least 13, 14, 15, 16, or 17 aminoacid (of 17) from VISPSGGRTLYADSVKG (SEQ ID NO:11); and/or (c) a CDR3that includes at least 5, 6, 7, or 8 amino acids (of 8) from HYSYAMDV(SEQ ID NO:12). In one embodiment, the protein includes features ofC1-54, e.g., the CDR regions of the C1-54 antibody. In one embodiment,the heavy and light chain variable domain sequences are at least 70, 80,85, 90, 92, 93, 94, 95, 97, 98, 99, or 100% identical to correspondingvariable domain sequences of the C1-54 antibody.

In another aspect, the disclosure features a protein that includes animmunoglobulin heavy chain (HC) variable domain sequence and animmunoglobulin light chain (LC) variable domain sequence, wherein the HCvariable domain sequence and the LC variable domain sequence form anantigen binding site that binds to an activated conformation of LFA-1,wherein the light chain variable domain sequence includes (a) a CDR1that includes at least 7, 8, 9, 10, or 11 amino acids (of 11) fromTASQSVDSNLA (SEQ ID NO:13), (b) a CDR2 that includes at least 4, 5, 6,or 7 amino acids (of 7) from GASTRAT (SEQ ID NO:14); and/or (c) a CDR3that includes at least 6, 7, 8, 9, or 10 amino acids (of 10) fromQQYNKWPPYS (SEQ ID NO:15). In one embodiment, the protein includesfeatures of C1-54, e.g., the CDR regions of the C1-54 antibody. In oneembodiment, the heavy and light chain variable domain sequences are atleast 70, 80, 85, 90, 92, 93, 94, 95, 97, 98, 99, or 100% identical tocorresponding variable domain sequences of the C1-54 antibody.

In another aspect, the disclosure features an antibody that includes animmunoglobulin heavy chain (HC) variable domain sequence and animmunoglobulin light chain (LC) variable domain sequence, wherein the HCvariable domain sequence and the LC variable domain sequence form anantigen binding site that binds to an activated conformation of LFA-1.The heavy chain variable domain sequence includes (a) a CDR1 thatincludes at least 3, 4, or 5 amino acids (of 5) from HYSMQ (SEQ IDNO:16), (b) a CDR2 that includes at least 13, 14, 15, 16, or 17 aminoacid (of 17) from YIGSSGGNTYYADSVKG (SEQ ID NO:17), and/or (c) a CDR3that includes at least 7, 8, 9, or 10 amino acids (of 10) fromGTYNTSPFDY (SEQ ID NO:18). In one embodiment, the protein includesfeatures of P1-G10, e.g., the CDR regions of the P1-010 antibody. In oneembodiment, the heavy and light chain variable domain sequences are atleast 70, 80, 85, 90, 92, 93, 94, 95, 97, 98, 99, or 100% identical tocorresponding variable domain sequences of the P1-G10 antibody.

In another aspect, the disclosure features a protein that includes animmunoglobulin heavy chain (HC) variable domain sequence and animmunoglobulin light chain (LC) variable domain sequence, wherein the HCvariable domain sequence and the LC variable domain sequence form anantigen binding site that binds to an activated conformation of LFA-1.The light chain variable domain sequence includes (a) a CDR1 thatincludes at least 7, 8, 9, 10, or 11 amino acids (of 11) fromSGDALGQKYAS (SEQ ID NO:19), (b) a CDR2 that includes at least 4, 5, 6,or 7 amino acids (of 7) from QDSKRPS (SEQ ID NO:20), and/or (c) a CDR3that includes at least 5, 6, 7, 8, or 9 amino acids (of 9) fromQAWDTTAYV (SEQ ID NO:21). In one embodiment, the protein includesfeatures of P1-G10, e.g., the CDR regions of the P1-G10 antibody. In oneembodiment, the heavy and light chain variable domain sequences are atleast 70, 80, 85, 90, 92, 93, 94, 95, 97, 98, 99, or 100% identical tocorresponding variable domain sequences of the P1-G10 antibody.

A protein described herein can have at least 30, 50, 60, 70, 80, 90 or100% of the CDR amino acid residues that are not identical to residuesin the reference CDR sequences be identical to residues at correspondingpositions in a human germline sequence. The protein can have at least30, 50, 60, 70, 80, 90 or 100% of the FR regions be identical to FRsequence from a human germline sequence or a FR sequence of D2-57,DX-2001, C1-54, or P1-G10. Exemplary human germline sequences includethose of VKI-O2, VL2-1, VKIII-L2::JK2, vg3-23, V3-23::JH4, andV3-23::JK6 and others provided herein.

In another aspect, the disclosure features an antibody or anon-naturally occurring protein that preferentially binds to activatedLFA-1 relative to inactivated LFA-1 and that competes with antibodyD2-57, DX-2001, C1-54, or P1-G10 for binding to activated LFA-1.

In another aspect, the disclosure features an antibody or anon-naturally occurring protein that binds to an epitope that overlapswith an epitope recognized by antibody D2-57, DX-2001, C1-54, or P1-G10on LFA-1, e.g., on activated LFA-1, or that binds to the same epitope asantibody D2-57, DX-2001, C1-54, or P1-G10.

In another aspect, the disclosure features a pharmaceutical compositionthat includes a protein described herein and a pharmaceuticallyacceptable salt. The invention also provides a kit that includes aprotein described herein and instructions for therapeutic or diagnosticuse.

In another aspect, the disclosure features a method of treating orpreventing inflammation or an inflammatory disorder. The methodincludes: administering a protein described herein to a subject in anamount effective to treat or prevent the inflammation or theinflammatory disorder, e.g., to ameliorate at least one symptom ofinflammation or the inflammatory disorder, or to delay the appearance ofsuch symptom.

In one embodiment, the protein is administered at dosages less than 1,0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, or 0.02 mg/kg per week, e.g.,for at least 2, 3, 5, 10, or 52 weeks. For example, the recommended dosefor the average patient can be less than 1, 0.7, 0.6, 0.5, 0.4, 0.3,0.2, 0.1, 0.05, or 0.02 mg/kg per week, e.g., for at least 2, 3, 5, 10,or 52 weeks.

In one embodiment, the protein is administered at dosages effective toproduce a detectable serum concentration whose mean trough concentrationis less than 9, 8, 7, 6, 5, 4, 3, 2, 1 μg/ml. In one embodiment, theprotein is administered in two phase, in which the first phase ischaracterized by administration of a first dose, and the second phase ischaracterized by administration of the second dose, different from thefirst dose. The first dose can be less than the second dose, or can begreater than the second dose, e.g., at least 20, 30, or 40% different.

For example, the first dose is an initial dose and, e.g., is less than0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, or 0.02 mg/kg. The second dosecan be less than 1, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, or 0.02mg/kg.

In one embodiment, the subject has psoriasis or is predisposed topsoriasis. For example, the subject has stable, plaque psoriasis. In oneembodiment, the subject has psoriasis whose minimum body surfaceinvolvement is at least 2, 5, 10, 15, 20, or 25%.

In one embodiment, the protein is administered to a subject who has notbeen treated with another systemic therapy or with phototherapy, e.g.,in the previous 30, 60, 90, or 180 days.

The protein can be administered at dosages effective to increase whiteblood cell count by at least 5, 10, 15, 20, 25, 30, 35, 40, or 45%. Theprotein can be administered at dosages effective to increase eosinophilscount by at least 5, 10, 15, 20, 25, 30, 35, 40, or 45%.

In another aspect, the disclosure features a method of treating orpreventing inflammation or an inflammatory disorder. The method includesadministering a protein described herein to a subject in an amounteffective to ameliorate inflammation or the inflammatory disorder,wherein the protein does not substantially interact with non-activatedLFA-1 molecules in the subject.

In one embodiment, the protein is administered at dosages less than 1,0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, or 0.02 mg/kg per week, e.g.,for at least 2, 3, 5, 10, or 52 weeks. For example, the recommended dosefor the average patient can be less than 1, 0.7, 0.6, 0.5, 0.4, 0.3,0.2, 0.1, 0.05, or 0.02 mg/kg per week, e.g., for at least 2, 3, 5, 10,or 52 weeks.

In one embodiment, the protein is administered at dosages effective toproduce a detectable serum concentration whose mean trough concentrationis less than 9, 8, 7, 6, 5, 4, 3, 2, 1 μg/ml. In one embodiment, theprotein is administered in two phase, in which the first phase ischaracterized by administration of a first dose, and the second phase ischaracterized by administration of the second dose, different from thefirst dose. The first dose can be less than the second dose, or can begreater than the second dose, e.g., at least 20, 30, or 40% different.

For example, the first dose is an initial dose and, e.g., is less than0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, or 0.02 mg/kg. The second dosecan be less than 1, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, or 0.02mg/kg.

In one embodiment, the subject has psoriasis or is predisposed topsoriasis. For example, the subject has stable, plaque psoriasis. In oneembodiment, the subject has psoriasis whose minimum body surfaceinvolvement is at least 2, 5, 10, 15, 20, or 25%.

In one embodiment, the protein is administered to a subject who has notbeen treated with another systemic therapy or with phototherapy, e.g.,in the previous 30, 60, 90, or 180 days.

The protein can be administered at dosages effective to increase whiteblood cell count by at least 5, 10, 15, 20, 25, 30, 35, 40, or 45%. Theprotein can be administered at dosages effective to increase eosinophilscount by at least 5, 10, 15, 20, 25, 30, 35, 40, or 45%.

In another aspect, the disclosure features a method of treating orpreventing an inflammation or an inflammatory disorder. The methodincludes: administering a protein described herein to a subject in anamount that is less than the amount required to treat or preventinflammation or the inflammatory disorder using an antibody that doesnot preferentially bind to activated LFA-1 (e.g., binds to bothactivated and inactivated LFA-1 with substantially the same affinity,e.g., RAPTIVA®), wherein the protein does not substantially interactwith non-activated LFA-1 molecules exposed on leukocytes of the subject.

In one embodiment, the protein is administered at dosages less than 1,0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, or 0.02 mg/kg per week, e.g.,for at least 2, 3, 5, 10, or 52 weeks. For example, the recommended dosefor the average patient can be less than 1, 0.7, 0.6, 0.5, 0.4, 0.3,0.2, 0.1, 0.05, or 0.02 mg/kg per week, e.g., for at least 2, 3, 5, 10,or 52 weeks. For example, the protein is administered at a dose lessthan RAPTIVA® to achieve substantially the same result.

In one embodiment, the protein is administered at dosages effective toproduce a detectable serum concentration whose mean trough concentrationis less than 9, 8, 7, 6, 5, 4, 3, 2, 1 μg/ml. In one embodiment, theprotein is administered in two phase, in which the first phase ischaracterized by administration of a first dose, and the second phase ischaracterized by administration of the second dose, different from thefirst dose. The first dose can be less than the second dose, or can begreater than the second dose, e.g., at least 20, 30, or 40% different.

For example, the first dose is an initial dose and, e.g., is less than0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, or 0.02 mg/kg. The second dosecan be less than 1, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, or 0.02mg/kg.

In one embodiment, the subject has psoriasis or is predisposed topsoriasis. For example, the subject has stable, plaque psoriasis. In oneembodiment, the subject has psoriasis whose minimum body surfaceinvolvement is at least 2, 5, 10, 15, 20, or 25%.

In one embodiment, the protein is administered to a subject who has notbeen treated with another systemic therapy or with phototherapy, e.g.,in the previous 30, 60, 90, or 180 days.

The protein can be administered at dosages effective to increase whiteblood cell count by at least 5, 10, 15, 20, 25, 30, 35, 40, or 45%. Theprotein can be administered at dosages effective to increase eosinophilscount by at least 5, 10, 15, 20, 25, 30, 35, 40, or 45%.

In another aspect, the disclosure features a method of treating orpreventing an inflammation or an inflammatory disorder. The methodincludes: administering a protein described herein to a subject in anamount effective to ameliorate or delay appearance of at least onesymptom of inflammation or the inflammatory disorder, wherein cells inthe subject that do not present an activated LFA-1 protein on theirsurface are not targeted by the protein.

In one embodiment, the protein is administered at dosages less than 1,0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, or 0.02 mg/kg per week, e.g.,for at least 2, 3, 5, 10, or 52 weeks. For example, the recommended dosefor the average patient can be less than 1, 0.7, 0.6, 0.5, 0.4, 0.3,0.2, 0.1, 0.05, or 0.02 mg/kg per week, e.g., for at least 2, 3, 5, 10,or 52 weeks.

In one embodiment, the protein is administered at dosages effective toproduce a detectable serum concentration whose mean trough concentrationis less than 9, 8, 7, 6, 5, 4, 3, 2, 1 μg/ml. In one embodiment, theprotein is administered in two phase, in which the first phase ischaracterized by administration of a first dose, and the second phase ischaracterized by administration of the second dose, different from thefirst dose. The first dose can be less than the second dose, or can begreater than the second dose, e.g., at least 20, 30, or 40% different.

For example, the first dose is an initial dose and, e.g., is less than0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, or 0.02 mg/kg. The second dosecan be less than 1, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, or 0.02mg/kg.

In one embodiment, the subject has psoriasis or is predisposed topsoriasis. For example, the subject has stable, plaque psoriasis. In oneembodiment, the subject has psoriasis whose minimum body surfaceinvolvement is at least 2, 5, 10, 15, 20, or 25%.

In one embodiment, the protein is administered to a subject who has notbeen treated with another systemic therapy or with phototherapy, e.g.,in the previous 30, 60, 90, or 180 days.

The protein can be administered at dosages effective to increase whiteblood cell count by at least 5, 10, 15, 20, 25, 30, 35, 40, or 45%. Theprotein can be administered at dosages effective to increase eosinophilscount by at least 5, 10, 15, 20, 25, 30, 35, 40, or 45%.

For example, the subject has or is predisposed to a disorder that iscaused at least in part by a T cell inflammatory response. In apreferred embodiment, the disorder is rheumatoid arthritis or psoriasis.

For example, the subject has or is predisposed to an inflammatorydisorder selected from the group consisting of: allergic conditions suchas eczema and asthma, Reiter's syndrome, HIV, cytokine-induced toxicity,transient hypogammaglobulinemia, malignancies (e.g., B-cell malignanciessuch as chronic lymphocytic leukemia or hairy cell leukemia), diseasesinvolving leukocyte diapedesis, acute glomerulonephritis, asthma, immunedeficiency disorders, invasion of tumor cells into secondary organsetc., insulinitis, atherosclerosis, conditions involving infiltration ofT cells and chronic inflammatory responses, selective IgA deficiency,meningitis, chronic mucocutaneous, dennatoses with acute inflammatorycomponents, sarcoidosis, skin hypersensitivity reactions (includingpoison ivy and poison oak), urticaria, nephrotic syndrome, acuteappendicitis, inflammatory bowel disease (such as Crohn's disease andulcerative colitis), encephalitis, wound healing, chronic obstructivepulmonary disease, myasthenia gravis, congenital X-linked infantilehypogammaglobulinemia, lupus, adult respiratory distress syndrome,orbital inflammatory disease, inflammatory breast disease, uveitis,psoriasis, HIV and rhinovirus infection, a CNS inflammatory disorder,antigen-antibody complex mediated diseases, necrotizing enterocolitis,amyloidosis, thermal injury, bronchitis, leukocyte adhesion deficiencyII syndrome, autoimmune hemolytic anemia, peritonitis, pulmonaryfibrosis, septic shock, multiple organ injury syndrome secondary tosepticemia or trauma, leukapheresis, pernicious anemia, nephritis,chronic bronchitis, common variable immunodeficiency, scleroderma,glomerulonephritis, polymyositis, pelvic inflammatory disease, rhinitis,granulocyte transfusion associated syndromes, ulcerative colitis andCrohns' disease), viral infection, hemodialysis, autoimmune diseases(e.g., granulomatosis and vasculitis), lung inflammation, reactivearthritis, dermatitis, and leukocyte adhesion deficiency. Example ofautoimmune disorders include: rheumatoid arthritis, systemic lupuserythematosus (SLE), diabetes mellitus, multiple sclerosis, Reynaud'ssyndrome, autoimmune thyroiditis, experimental autoimmuneencephalomyelitis, Sjorgen's syndrome, juvenile onset diabetes, andimmune responses associated with delayed hypersensitivity mediated bycytokines and T-lymphocytes typically found in tuberculosis,sarcoidosis, and polymyositis,

In one embodiment, the protein is administered at a dosage that does notsubstantially increase risk for serious infection (e.g., no more than0.4% of patients), risk for thrombocytopenia(e.g., no more than 0.3% ofpatients), risk for psoriasis aggravation (e.g., no more than 0.7% ofpatients), or risk or headache, chill, fever, nausea, myalgia, pain,arthritis, or arthralgia (e.g., no more than 32, 13, 7, 11, 8, 10, 0.4,and 0.3% of patients, respectively). In one embodiment, the protein canthe same frequency of side effects as RAPTIVA®, or less.

In another aspect, the disclosure features a method of suppressing animmune response. The method includes a protein described herein to asubject in an amount effective to suppress an immune response of thesubject. In one embodiment, the subject has or is about to receive atransplant.

In another aspect, the disclosure features a method of treating orpreventing a disorder in a subject. The method includes: identifying asubject in need of an anti-LFA-1 antibody that preferentially binds tothe activated form of LFA-1, but which subject does not respond ortolerate an anti-LFA-1 antibody that binds to activated andnon-activated LFA-1 protein with substantially the same affinity; andadministering the anti-LFA-1 antibody that preferentially binds to theactivated form of LFA-1, to the subject.

In another aspect, the disclosure features a method of modulating aLFA-1activity. The method includes: providing an aLFA-1-binding protein ofclaim 1; and contacting the protein to aLFA-1, in an amount sufficientto modulate aLFA-1 activity.

For example, the contacting is in vitro or in vivo.

In one embodiment, the protein is contacted to aLFA-1 in the vicinity ofa neoplastic cell (e.g., a cell found in laryngeal, epidermal,pulmonary, breast, renal, urothelial, colonic, prostatic, or hepaticcancer and/or metastasis). In one embodiment, the protein is contactedto aLFA-1 in the vicinity of an endothelial cell.

In another aspect, the disclosure features a method for detecting thepresence of an aLFA-1 protein, in a sample, e.g., in vitro. The methodincludes: (i) contacting the sample (and optionally, a reference, e.g.,control, sample) with an aLFA-1-binding protein described herein, underconditions that allow interaction of the aLFA-1-binding protein and theaLFA-1 protein to occur; and (ii) detecting interaction between theaLFA-1-binding protein, and the sample (and optionally, the reference,e.g., control, sample).

At least one of the aLFA-1 binding protein or the aLFA-1 is immobilized.

In another aspect, the disclosure features a method for detecting thepresence of aLFA-1 (e.g., activated aLFA-1), e.g., in vivo. The methodincludes: (i) administering to a subject (and optionally a controlsubject) an aLFA-1-binding protein, under conditions that allowinteraction of the aLFA-1-binding protein and the aLFA-1 protein tooccur; and (ii) detecting location of the aLFA-1-binding protein in thesubject or formation of a complex between the aLFA-1-binding protein andaLFA-1 in the subject. For example, the subject is a human subject. Thedetecting can include imaging the subject. For example, theaLFA-1-binding protein is labeled with an MRI detectable label.

The invention also includes a protein that includes an immunoglobulinheavy chain (HC) variable domain sequence and an immunoglobulin lightchain (LC) variable domain sequence The HC variable domain sequence andthe LC variable domain sequence form an antigen binding site thatdetectably binds to both an integrin I-domain in the activatedconformation and an integrin I-domain in the non-activated conformation,but preferentially binds to an integrin in the activated conformationrelative to binding to the integrin in the non-activated conformation.

For example, the protein has at least a 1.5, 2, 3, 4, 5, 10, 15, 20, 50,70, 80, 100, 500, or 1000 fold preference for binding to activated LFA-1relative to inactivated LFA-1.

The protein can have at least a 1.5, 2, 3, 4, 5, or 10 preference forbinding to activated LFA-1 relative to inactivated LFA-1, but no morethan a 15, 20, 50, 70, 80, 100, 500, or 1000 fold preference.

The invention also includes a protein that includes an immunoglobulinheavy chain (HC) variable domain sequence and an immunoglobulin lightchain (LC) variable domain sequence. The HC variable domain sequence andthe LC variable domain sequence form an antigen binding site thatdetectably binds to both an integrin I-domain in the open conformationand an integrin I-domain in the closed conformation, but preferentiallybinds to the integrin I-domain in the open conformation relative to theintegrin I-domain in the closed conformation.

For example, the protein has at least a 1.5, 2, 3, 4, 5, 10, 15, 20, 50,70, 80, 100, 500, or 1000 fold preference for binding to activated LFA-1relative to inactivated LFA-1.

The protein can have at least a 1.5, 2, 3, 4, 5, or 10 preference forbinding to activated LFA-1 relative to inactivated LFA-1, but no morethan a 15, 20, 50, 70, 80, 100, 500, or 1000 fold preference. In oneembodiment, protein can bind to a disulfide-locked K287C/K294C I-domain.

The invention also includes a protein that includes an immunoglobulinheavy chain (HC) variable domain sequence and an immunoglobulin lightchain (LC) variable domain sequence, wherein the HC variable domainsequence and the LC variable domain sequence form an antigen bindingsite that detectably binds to both an integrin I-domain of LFA-1 in theactivated conformation and an integrin I-domain in the non-activatedconformation, but preferentially binds to activated LFA-1 relative tonon-activated LFA-1. For example, the protein has at least a 1.5, 2, 3,4, 5, 10, 15, 20, 50, 70, 80, 100, 500, or 1000 fold preference forbinding to activated LFA-1 relative to inactivated LFA-1. The proteincan have at least a 1.5, 2, 3, 4, 5, or 10 preference for binding toactivated LFA-1 relative to inactivated LFA-1, but no more than a 15,20, 50, 70, 80, 100, 500, or 1000 fold preference. In one embodiment,the 1-domain in the open conformation is a disulfide-locked K287C/K294CI-domain.

Exemplary antibodies can include the following sequences or segmentsthereof:

TABLE 1 Exemplary Variable Domains Name Amino Acid Sequence D2-57 LCDIQMTQSPSSLSASVGRVTITC RASQSIGSYLN SEQ ID NO: 22WYQQKKTGKAPKALIY AASSLQS GVPSRFSGSGSGTDFTLTISSLQLEDFATYYCQQSYSTPS FGQGTKVEIKRT D2-57 HC EVQLLESGGGLVQPGGSLRLSCAASGFTFS RYVMWSEQ ID NO: 23 WVRQAPGKLGLEWVS YIWPSGGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAS STDFWSNAFDI WGQGTMVTVSS DX-2001 LCDIQMTQSPSSLSASVGDRVTITC RASQSIGSYLN SEQ ID NO: 24WYQQKPGKAPKALIY AASSLQS GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPSFGQGTKVEIKRT DX-2001 HC EVQLLESGGGLVQPGGSLRLSCAASGFTFS RYVMWSEQ ID NO: 25 WVRQAPFKGLEWVS YIWPSGGNTYYADSVKGRFTISRDNSKNTLYLQMNLSRAEDTAVYYCAS SYDFWSNAFDIWGQGTMVTVSS C1-54 LCDIQMTQSPATLSVSPGERVTLSC TASWSVDSNLA SEQ ID NO: 26WYQQKPGQAPRLLVY GASTRAT GVPARFSGSGSGTAFTLTIDSLQSEDFAVYYCQQYNKWPPYS FGQGTKLEIKRT C1-54 HC EVQLLESGGGLVQPGGSLRLSCAASGFTFS HYGMSSEQ ID NO: 27 WVRQAPGKGLEWVW VISPSGGRTLYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK HYSYAMDV WGQGTTVTVSS P1-G10 LCSVLTQPPSVSVSPGQTASVTC SGDALGQKYAS SEQ ID NO: 28 WYQQKPGQSPVLVIF QDSKRPSGIPERFSGSNSGNTATLTISGTQAVDEADYYC QAWDTTAYV FGTGTKVTVL P1-G10 HCEVQLLESGGGLVQPGGSLRLSCAASGFTFS HYSMQ SEQ ID NO: 29WVRQAPGKGLEWVS YIGSSGGNTYYADSVKG RFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGTYNYSPFDY WGQGTLVTVSS Versions of the heavy chain variable domain canomit, e.g., the N-terminal glutamic acid.

An integrin binding antibody is typically monospecific, e.g., amonoclonal antibody, or antigen-binding fragment thereof. TheaLFA-1-binding antibodies can be full-length (e.g., an IgG (e.g., anIgG1, IgG2, IgG3, IgG4), IgM, IgA (e.g., IgA1, IgA2), IgD, and IgE) orcan include only an antigen-binding fragment (e.g., a Fab, F(ab′)₂ orscFv fragment). The antibody, or antigen-binding fragment thereof, caninclude two heavy chain immunoglobulins and two light chainimmunoglobulins, or can be a single chain antibody. The antibodies can,optionally, include a constant region chosen from a kappa, lambda,alpha, gamma, delta, epsilon or a mu constant region gene. AnaLFA-1-binding antibody can include a heavy and light chain constantregion substantially from a human antibody, e.g., a human IgG1 IgG2,IgG3, or IgG4 constant region or a portion thereof. The constant regioncan have the sequence of an A or non-A allotype.

In one embodiment, the antibody is a recombinant or modified antibody,e.g., a chimeric, a humanized, a deimmunized, or an in vitro generatedantibody. The term “recombinant” or “modified” antibody, as used herein,is intended to include all antibodies that are prepared, expressed,created or isolated by recombinant means, such as antibodies expressedusing a recombinant expression vector transfected into a host cell,antibodies isolated from a recombinant, combinatorial antibody library,antibodies isolated from an animal (e.g., a mouse) that is transgenicfor human immunoglobulin genes or antibodies prepared, expressed,created or isolated by any other means that involves splicing of humanimmunoglobulin gene sequences to other DNA sequences. Such recombinantantibodies include human, humanized, CDR grafted, chimeric, deimmunized,in vitro generated antibodies, and may optionally include frameworkand/or constant regions derived from human germlineimmunoglobulin-encoding nucleic acid sequences.

In one embodiment, the antibody binds to an epitope distinct from anepitope bound by known antibodies that bind to LFA-1. For example, theantibody binds to an epitope that distinct from one or more of theepitopes bound by MHM23 (Hildreth et al., Eur. J. Immunol. 13:202-208(1983)); MHM24; RAPTIVA®; M18/2 (IgG.sub.2a; Sanches-Madrid et al., J.Exp. Med. 158:586 (1983)); mAb25 (Dranfield et al, J. Cell Biol. 1992January; 116(1):219-26.) H52 (American Type Culture Collection (ATCC)Deposit HB 10160); NKI-L16 (Landis et al., J. Cell Biol. 1993 March;120(6):1519-27); MEM-83 (Landis et al., supra); 7E3; Mas191c and IOT18(Vermot Desroches et al., Scand. J. Immunol. 33:277-286 (1991)); andNA-8 (WO 94/12214). In other embodiments, the antibody does not competewith such antibodies for bind to LFA-1. In still other embodiments, theantibody does not compete with an antibody described herein.

In one embodiment, the antibody binds to overlapping epitopes of, orcompetitively inhibits, the binding of an antibody disclosed herein toaLFA-1, e.g., D2-57, DX-2001, C1-54, or P1-G10. In one embodiment, theantibody binds to an epitope that includes an amino acid that is withinat least 12, 10, 8, 6, 5, or 3 amino acids of an epitope bound by anantibody described herein (e.g., D2-57, DX-2001, C1-54, or P1-G10). Inone embodiment, the antibody includes an antigen binding site structurethat recognizes one or more side chains that are positioned within 12,10, 8, 6 or 4 Angstroms of an antibody described herein (e.g., D2-57,DX-2001, C1-54, or P1-G10). The epitope is generally in theextracellular region of LFA-1. The epitope can include one or more aminoacid side chains on the α and/or β subunit. In one embodiment, theepitope includes one or more amino acid side chains on the I-domain ofαL.

Further, any combination of aLFA-1-binding antibodies is within thescope of the invention, e.g., two or more antibodies that bind todifferent regions of aLFA-1, e.g., antibodies that bind to two differentepitopes on the extracellular domain of aLFA-1, e.g., a bispecificantibody.

In one embodiment, the aLFA-1-binding antibody includes at least onelight or heavy chain immunoglobulin (or two light chain immunoglobulinsand two heavy chain immunoglobulins). Preferably, each immunoglobulinincludes a light or a heavy chain variable region having at least one,two and, preferably, three complementarity determining regions (CDRs)substantially identical to a CDR from a light or heavy chain variableregion, respectively, of an antibody described herein.

An integrin binding protein described herein can be used alone, e.g.,can be administered to a subject or used in vitro in non-derivatized orunconjugated forms. In other embodiments, the integrin binding proteincan be derivatized, modified or linked to another functional molecule,e.g., another polypeptide, protein, isotope, cell, or insoluble support.For example, the integrin binding protein can be functionally linked(e.g., by chemical coupling, genetic fusion, non-covalent association orotherwise) to one or more other molecular entities, such as an antibody(e.g., if the binding protein is an antibody, to form a bispecific or amulti-specific antibody), a toxin, a label, a serum-residence prolongingmoiety (e.g. PEG), a therapeutic (e.g., a cytotoxic or cytostatic) agentor other moiety. An antibody can also be designed so that it can mediatecomplement-dependent cytotoxicity (CDC) or antibody-dependent cellularcytotoxicity (ADCC), or so that it does not mediate CDC or ADCC. Forexample, it can have a CDC- or ADCC-competent Fc domain, or a CDC- orADCC-incompetent Fc domain.

In another aspect, the disclosure features a nucleic acid that includesa coding sequence that encodes a polypeptide including an immunoglobulinheavy chain variable domain sequence that binds to aLFA-1, e.g., animmunoglobulin heavy chain variable domain described herein. Forexample, the immunoglobulin heavy chain variable domain sequence caninclude: a CDR motif or CDR described herein. The immunoglobulin heavychain variable domain sequence can include a framework region describedherein. In one example, the variable domain sequence is a heavy chainvariable domain is at least 75, 80, 85, 90, 95, 96, 97, 98, or 99%identical to an amino acid sequence described herein or a variabledomain sequence thereof.

In another aspect, the disclosure features a nucleic acid that includesa coding sequence that encodes a polypeptide including an immunoglobulinlight chain variable domain sequence that binds to aLFA-1, e.g., animmunoglobulin light chain variable domain described herein. Forexample, the immunoglobulin light chain variable domain sequence caninclude: a CDR motif or CDR described herein. The immunoglobulin lightchain variable domain sequence can include a framework region describedherein. In one example, the variable domain sequence is a light chainvariable domain is at least 75, 80, 85, 90, 95, 96, 97, 98, or 99%identical to an amino acid sequence described herein or a variabledomain sequence thereof.

A nucleic acid described herein can further include a promoter operablylinked to the coding sequence. A nucleic acid can include a first andsecond coding sequence, e.g., wherein the first coding sequence encodesa polypeptide that includes an immunoglobulin heavy chain variabledomain and the second coding sequence encodes a polypeptide thatincludes an immunoglobulin light chain variable domain.

In another aspect, the disclosure features a host cell that contains afirst nucleic acid encoding a polypeptide including a heavy chainvariable region and a second nucleic acid encoding a polypeptideincluding a light chain variable region. The heavy chain variable regionand the light chain variable region can associate to form an aLFA-1binding protein. These variable regions can have one or more propertiesdescribed herein, e.g., at least 75, 80, 85, 90, 95, 96, 97, 98, or 99%identity to a sequence described herein, e.g., the sequence of avariable domain from an isolated antibody described herein or a humangermline sequence described herein. The invention also includes a methodof providing an aLFA-1-binding antibody. The method can includeproviding a host cell described herein; and expressing said first andsecond nucleic acids in the host cell under conditions that allowassembly of said light and heavy chain variable regions to form anantigen binding protein that interacts with aLFA-1.

In another aspect, the disclosure features a binding protein thatincludes a human or effectively human heavy chain immunoglobulinvariable domain and a human or effectively human light chainimmunoglobulin variable domain, wherein the binding protein binds tohuman aLFA-1. The protein can bind to aLFA-1 with a K_(d) of less than,10⁻⁷, 10⁻⁸, 10⁻⁹, or 10⁻¹⁰ M. The protein can include one or moreadditional features described herein.

In yet another aspect, the disclosure features a method of producing anaLFA-1-binding antibody, or antigen-binding fragment thereof. The methodincludes: providing a host cell that contains a first nucleic acidsequence encoding a polypeptide including a heavy chain variable region,e.g., a heavy chain variable region as described herein; providing asecond nucleic acid sequence encoding a polypeptide including a lightchain variable region, e.g., a light chain variable region as describedherein; and expressing said first and second nucleic acid sequences inthe host cell under conditions that allow assembly of said light andheavy chain variable regions to form an antigen binding protein thatinteracts with aLFA-1. The first and second nucleic acid sequences canbe linked or unlinked, e.g., expressed on the same or different vector,respectively. The first and second nucleic acid sequences can becomponents of the same molecule or can reside on different molecules(e.g., different chromosomes or plasmids).

The host cell can be a eukaryotic cell, e.g., a mammalian cell, aninsect cell, a yeast cell, or a prokaryotic cell, e.g., E. coli. Forexample, the mammalian cell can be a cultured cell or a cell line.Exemplary mammalian cells include lymphocytic cell lines (e.g., NSO),Chinese hamster ovary cells (CHO), COS cells, oocyte cells, and cellsfrom a transgenic animal, e.g., mammary epithelial cell. For example,nucleic acids encoding the antibodies described herein can be expressedin a transgenic animal. In one embodiment, the nucleic acids are placedunder the control of a tissue-specific promoter (e.g., a mammaryspecific promoter) and the antibody is produced in the transgenicanimal. For example, the antibody molecule is secreted into the milk ofthe transgenic animal, such as a transgenic cow, pig, horse, sheep, goator rodent.

In another aspect, the disclosure features a method of treating orpreventing an inflammatory disorder in a subject. The method includesproviding an aLFA-1-binding protein, e.g. a protein described herein,and contacting the subject with the protein, in an amount sufficient tomodulate or prevent the inflammatory disorder. The method can includeidentifying a subject as having or being at risk for having aninflammatory disorder.

The subject can be a mammal, e.g., a primate, preferably a higherprimate, e.g., a human (e.g., a patient having, or at risk of, adisorder described herein).

The aLFA-1-binding protein can be administered to the subjectsystemically (e.g., orally, parenterally, subcutaneously, intravenously,intramuscularly, intraperitoneally, intranasally, transdermally, or byinhalation), topically, or by application to mucous membranes, such asthe nose, throat and bronchial tubes.

The method can further include monitoring at least one indicator ofinflammation, e.g., local temperature, swelling (e.g., as measured),redness, local or systemic white blood cell count, presence or absenceof neutrophils, cytokine levels, elastase activity, and so forth. Thesubject can be monitored in one or more of the following periods: priorto beginning of treatment; during the treatment; or after one or moreelements of the treatment have been administered. Monitoring can be usedto evaluate the need for further treatment with the same aLFA-1-bindingprotein or other agents. A desired change in one or more of theparameters described above can be indicative of the improved conditionof the subject. Information about the monitoring can be recorded, e.g.,in electronic or digital form.

In another aspect, the disclosure features methods for detecting thepresence of an aLFA-1 protein, in a sample, in vitro (e.g., a biologicalsample or a tissue biopsy). The subject method can be used to evaluate,e.g., diagnose or stage a disorder described herein. The methodincludes: (i) contacting the sample (and optionally, a reference, e.g.,control, sample) with an aLFA-1-binding protein, as described herein,under conditions that allow interaction of the aLFA-1-binding proteinand the LFA-1 protein to occur; and (ii) detecting aLFA-1, e.g., bydetecting formation of a complex between the LFA-1-binding protein andLFA-1, or by detecting an interaction between the aLFA-1-binding proteinand LFA-1, in the sample (and optionally, the reference, e.g., control,sample). Formation of the complex can be indicative of the presence ofaLFA-1 protein (e.g., activated aLFA-1 protein), and can indicate thesuitability or need for a treatment described herein. For example, astatistically significant change in the formation of the complex in thesample relative to the reference sample, e.g., the control sample, isindicative of the presence of activated aLFA-1 in the sample.

In yet another aspect, the invention provides a method for detecting thepresence of LFA-1 (e.g., activated aLFA-1) in vivo (e.g., in vivoimaging in a subject). The subject method can be used to evaluate, e.g.,diagnose, localize, or stage a disorder described herein, e.g.,inflammation, an inflammatory disorder, a disorder characterized byexcessive LFA-1 activity, or a LFA-1 mediated disorder. The methodincludes:

-   (i) administering to a subject (and optionally a control subject) an    aLFA-1-binding protein (e.g., an antibody or antigen binding    fragment thereof), under conditions that allow interaction of the    aLFA-1-binding protein and the aLFA-1 protein to occur; and-   (ii) detecting formation of a complex between the binding protein    and aLFA-1, wherein a statistically significant change in the    formation of the complex in the subject relative to the reference,    e.g., the control subject or subject's baseline, is indicative of    the presence of the aLFA-1. The presence of activated aLFA-1 in    particular locations within a subject can be indicative of    inflammation or an inflammatory disorder.

In other embodiments, a method of diagnosing or staging, a disorder asdescribed herein (e.g., an inflammatory disorder, a disordercharacterized by excessive LFA-1 activity, or a LFA-1 mediateddisorder), is provided. The method includes: (i) identifying a subjecthaving, or at risk of having, the disorder; (ii) obtaining a sample of atissue or cell affected with the disorder; (iii) contacting said sampleor a control sample with an aLFA-1-binding protein, under conditionsthat allow interaction of the binding agent and the aLFA-1 protein tooccur, and (iv) detecting formation of a complex. A statisticallysignificant increase in the formation of the complex between the bindingprotein and LFA-1 with respect to a reference sample, e.g., a controlsample, is indicative of the disorder or the stage of the disorder. Inone embodiment, the sample is obtained by non-surgical means, e.g., by ablood, saliva, or urine sample. In another embodiment, surgery is used.

Preferably, the aLFA-1-binding protein used in the in vivo and in vitrodiagnostic methods is directly or indirectly labeled with a detectablesubstance to facilitate detection of the bound or unbound binding agent.

Although many embodiments of the disclosure are described in the contextof binding proteins that preferentially bind to activated LFA-1(“aLFA-1”), proteins that preferentially bind to a conformer of anothertarget protein (e.g., another integrin, e.g., another leukocyte integrinsubfamily member) or a different LFA-1 conformer can also be made andused.

Definitions

The term “binding protein” refers to a protein that can interact with atarget molecule. An “integrin binding protein” refers to a protein thatcan interact with an integrin, and includes, in particular proteins thatpreferentially interact with an activated integrin, e.g., aLFA-1, ormimic thereof.

As used herein, the term “antibody” refers to a protein that includes atleast one immunoglobulin variable domain or immunoglobulin variabledomain sequence. For example, an antibody can include a heavy (H) chainvariable region (abbreviated herein as VH), and a light (L) chainvariable region (abbreviated herein as VL). In another example, anantibody includes two heavy (H) chain variable regions and two light (L)chain variable regions. The term “antibody” encompasses antigen-bindingfragments of antibodies (e.g., single chain antibodies, Fab fragments,F(ab′)₂, a Fd fragment, a Fv fragments, and dAb fragments) as well ascomplete antibodies.

The VH and VL regions can be further subdivided into regions ofhypervariability, termed “complementarity determining regions” (“CDR”),interspersed with regions that are more conserved, termed“frameworkregions” (FR). The extent of the framework region and CDRs has beenprecisely defined (see, Kabat, et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242, and Chothia, C. et al.(1987) J. Mol. Biol. 196:901-917). Kabat definitions are used herein.Each VH and VL is typically composed of three CDRs and four FRs,arranged from amino-terminus to carboxy-terminus in the following order:FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

An “immunoglobulin domain” refers to a domain from the variable orconstant domain of immunoglobulin molecules. Immunoglobulin domainstypically contain two β-sheets formed of about seven β-strands, and aconserved disulphide bond (see, e.g., A. F. Williams and A. N. Barclay1988 Ann. Rev Immunol. 6:381-405). The canonical structures ofhypervariable loops of an immunoglobulin variable domain can be inferredfrom its sequence, e.g., as described in Chothia et al. (1992) J. Mol.Biol. 227:799-817; Tomlinson et al. (1992) J. Mol. Biol. 227:776-798);and Tomlinson et al. (1995) EMBO J. 14(18):4628-38.

As used herein, an “immunoglobulin variable domain sequence” refers toan amino acid sequence which can form the structure of an immunoglobulinvariable domain. For example, the sequence may include all or part ofthe amino acid sequence of a naturally-occurring variable domain. Forexample, the sequence may omit one, two or more N- or C-terminal aminoacids, internal amino acids, may include one or more insertions oradditional terminal amino acids, or may include other alterations. Inone embodiment, a polypeptide that includes immunoglobulin variabledomain sequence can associate with another immunoglobulin variabledomain sequence to form a target binding structure (or “antigen bindingsite”), e.g., a structure that preferentially interacts with anactivated integrin structure or a mimic of an activated integrinstructure, e.g., relative to an non-activated structure.

The VH or VL chain of the antibody can further include all or part of aheavy or light chain constant region, to thereby form a heavy or lightimmunoglobulin chain, respectively. In one embodiment, the antibody is atetramer of two heavy immunoglobulin chains and two light immunoglobulinchains, wherein the heavy and light immunoglobulin chains areinter-connected by, e.g., disulfide bonds. The heavy chain constantregion includes three domains, CH1, CH2 and CH3. The light chainconstant region includes a CL domain. The variable region of the heavyand light chains contains a binding domain that interacts with anantigen. The constant regions of the antibodies typically mediate thebinding of the antibody to host tissues or factors, including variouscells of the immune system (e.g., effector cells) and the firstcomponent (Clq) of the classical complement system. The term “antibody”includes intact immunoglobulins of types IgA, IgG, IgE, IgD, IgM (aswell as subtypes thereof). The light chains of the immunoglobulin may beof types kappa or lambda. In one embodiment, the antibody isglycosylated. An antibody can be functional for antibody-dependentcytotoxicity and/or complement-mediated cytotoxicity.

One or more regions of an antibody can be human or effectively human.For example, one or more of the variable regions can be human oreffectively human. For example, one or more of the CDRs can be human,e.g., HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2, and LC CDR3. Each ofthe light chain CDRs can be human: HC CDR3 can be human. One or more ofthe framework regions can be human, e.g., FR1, FR2, FR3, and FR4 of theHC or LC. In one embodiment, all the framework regions are human, e.g.,derived from a human somatic cell, e.g., a hematopoietic cell thatproduces immunoglobulins or a non-hematopoietic cell. In one embodiment,the human sequences are germline sequences, e.g., encoded by a germlinenucleic acid. One or more of the constant regions can be human oreffectively human. In another embodiment, at least 70, 75, 80, 85, 90,92, 95, or 98% of, or the entire antibody can be human or effectivelyhuman.

All or part of an antibody can be encoded by an immunoglobulin gene or asegment thereof. Exemplary human immunoglobulin genes include the kappa,lambda, alpha (IgA1 and IgA2), gamma (IgG1, IgG2, IgG3, IgG4), delta,epsilon and mu constant region genes, as well as the myriadimmunoglobulin variable region genes. Full-length immunoglobulin “lightchains” (about 25 Kd or 214 amino acids) are encoded by a variableregion gene at the NH2-terminus (about 110 amino acids) and a kappa orlambda constant region gene at the carboxy terminus. Full-lengthimmunoglobulin “heavy chains” (about 50 Kd or 446 amino acids), aresimilarly encoded by a variable region gene (about 116 amino acids) andone of the other aforementioned constant region genes, e.g., gamma(encoding about 330 amino acids).

The term “antigen-binding fragment” of a full length antibody (or simply“antibody portion,” or “fragment”), as used herein, refers to one ormore fragments of a full-length antibody that retain the ability tospecifically bind to a target of interest. Examples of binding fragmentsencompassed within the term “antigen-binding fragment” of a full lengthantibody include (i) a Fab fragment, a monovalent fragment consisting ofthe VL, VH, CL and CH1 domains; (ii) a F(ab′)₂ fragment, a bivalentfragment including two Fab fragments linked by a disulfide bridge at thehinge region; (iii) a Fd fragment consisting of the VH and CH1 domains;(iv) a Fv fragment consisting of the VL and VH domains of a single armof an antibody, (v) a dAb fragment (Ward et al., (1989) Nature341:544-546), which consists of a VH domain; and (vi) an isolatedcomplementarity determining region (CDR) that retains functionality.Furthermore, although the two domains of the Fv fragment, VL and VH, arecoded for by separate genes, they can be joined, using recombinantmethods, by a synthetic linker that enables them to be made as a singleprotein chain in which the VL and VH regions pair to form monovalentmolecules known as single chain Fv (scFv). See e.g., Bird et al. (1988)Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA85:5879-5883.

Antibody fragments can be obtained using any appropriate techniqueincluding conventional techniques known to those with skill in the art.The term “monospecific antibody” refers to an antibody that displays asingle binding specificity and affinity for a particular target, e.g.,epitope. This term includes a “monoclonal antibody” or “monoclonalantibody composition,” which as used herein refer to a preparation ofantibodies or fragments thereof of single molecular composition. As usedherein, “isotype” refers to the antibody class (e.g., IgM or IgG1) thatis encoded by heavy chain constant region genes.

An “effectively human” immunoglobulin variable region is animmunoglobulin variable region that includes a sufficient number ofhuman framework amino acid positions such that the immunoglobulinvariable region does not elicit an immunogenic response in a normalhuman. An “effectively human” antibody is an antibody that includes asufficient number of human amino acid positions such that the antibodydoes not elicit an immunogenic response in a normal human.

A “humanized” immunoglobulin variable region is an immunoglobulinvariable region that is modified to include a sufficient number of humanframework amino acid positions such that the immunoglobulin variableregion does not elicit an immunogenic response in a normal human.Descriptions of “humanized” immunoglobulins include, for example, U.S.Pat. Nos. 6,407,213 and 5,693,762.

As used herein, “binding affinity” refers to the apparent associationconstant or K_(a). The K_(a) is the reciprocal of the dissociationconstant (K_(d)). A binding protein may, for example, have a bindingaffinity of at least 10⁻⁵, 10⁻⁶, 10⁻⁷ or 10⁻⁸ M for a particular targetmolecule. Higher affinity binding of a binding ligand to a first targetrelative to a second target can be indicated by a higher K_(a) (or asmaller numerical value K_(d)) for binding the first target than theK_(a) (or numerical value K_(d)) for binding the second target. In suchcases the binding protein has specificity for the first target (e.g., aprotein in a first conformation or mimic thereof) relative to the secondtarget (e.g., the same protein in a second conformation or mimicthereof). Differences in binding affinity (e.g., for specificity orother comparisons) can be at least 1.5, 2, 3, 4, 5, 10, 15, 20, 50, 70,80, 100, 500, 1000, or 10⁵ fold.

Binding affinity can be determined by a variety of methods includingequilibrium dialysis, equilibrium binding, gel filtration, ELISA,surface plasmon resonance, or spectroscopy (e.g., using a fluorescenceassay). Exemplary conditions for evaluating binding affinity are in PBS(phosphate buffered saline) at pH 7.2 at 30° C. These techniques can beused to measure the concentration of bound and free binding protein as afunction of binding protein (or target) concentration. The concentrationof bound binding protein ([Bound]) is related to the concentration offree binding protein ([Free]) and the concentration of binding sites forthe binding protein on the target where (N) is the number of bindingsites per target molecule by the following equation:[Bound]=N·[Free]/((1/Ka)+[Free]).

It is not always necessary to make an exact determination of Ka, though,since sometimes it is sufficient to obtain a quantitative measurement ofaffinity, e.g., determined using a method such as ELISA or FACSanalysis, is proportional to Ka, and thus can be used for comparisons,such as determining whether a higher affinity is, e.g., 2-fold higher,to obtain a qualitative measurement of affinity, or to obtain aninference of affinity, e.g., by activity in a functional assay, e.g., anin vitro or in vivo assay.

An “isolated composition” refers to a composition that is removed fromat least 90% of at least one component of a natural sample from whichthe isolated composition can be obtained. Compositions producedartificially or naturally can be “compositions of at least” a certaindegree of purity if the species or population of species of interests isat least 5, 10, 25, 50, 75, 80, 90, 92, 95, 98, or 99% pure on aweight-weight basis.

An “epitope” refers to the site on a target compound that is bound by abinding protein (e.g., an antibody such as a Fab or full lengthantibody). In the case where the target compound is a protein, the sitecan be entirely composed of amino acid components, entirely composed ofchemical modifications of amino acids of the protein (e.g., glycosylmoieties), or composed of combinations thereof. Overlapping epitopesinclude at least one common amino acid residue.

Calculations of “homology” or “sequence identity” between two sequences(the terms are used interchangeably herein) are performed as follows.The sequences are aligned for optimal comparison purposes (e.g., gapscan be introduced in one or both of a first and a second amino acid ornucleic acid sequence for optimal alignment and non-homologous sequencescan be disregarded for comparison purposes). The optimal alignment isdetermined as the best score using the GAP program in the GCG softwarepackage with a Blossum 62 scoring matrix with a gap penalty of 12, a gapextend penalty of 4, and a frameshift gap penalty of 5. The amino acidresidues or nucleotides at corresponding amino acid positions ornucleotide positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences.

In a preferred embodiment, the length of a reference sequence alignedfor comparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, even more preferably at least 60%, and evenmore preferably at least 70%, 80%, 90%, 92%, 95%, 97%, 98%, or 100% ofthe length of the reference sequence. For example, the referencesequence may be the length of the immunoglobulin variable domainsequence.

As used herein, the term “substantially identical” (or “substantiallyhomologous”) is used herein to refer to a first amino acid or nucleicacid sequence that contains a sufficient number of identical orequivalent (e.g., with a similar side chain, e.g., conserved amino acidsubstitutions) amino acid residues or nucleotides to a second amino acidor nucleic acid sequence such that the first and second amino acid ornucleic acid sequences have (or encode proteins having) similaractivities, e.g., a binding activity, a binding preference, or abiological activity. In the case of antibodies, the second antibody hasthe same specificity and has at least 50% of the affinity relative tothe same antigen.

Sequences similar or homologous (e.g., at least about 85% sequenceidentity) to the sequences disclosed herein are also part of thisapplication. In some embodiment, the sequence identity can be about 85%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. In addition,substantial identity exists, e.g., when the nucleic acid segmentshybridize under selective hybridization conditions (e.g., highlystringent hybridization conditions), to the complement of the strand.The nucleic acids may be present in whole cells, in a cell lysate, or ina partially purified or substantially pure form.

As used herein, the term “hybridizes under low stringency, mediumstringency, high stringency, or very high stringency conditions”describes conditions for hybridization and washing. Guidance forperforming hybridization reactions can be found in Current Protocols inMolecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6, which isincorporated by reference. Aqueous and nonaqueous methods are describedin that reference and either can be used. Specific hybridizationconditions referred to herein are as follows: (1) low stringencyhybridization conditions in 6× sodium chloride/sodium citrate (SSC) atabout 45° C., followed by two washes in 0.2×SSC, 0.1% SDS at least at50° C. (the temperature of the washes can be increased to 55° C. for lowstringency conditions); (2) medium stringency hybridization conditionsin 6×SSC at about 45° C., followed by one or more washes in 0.2×SSC,0.1% SDS at 60° C.; (3) high stringency hybridization conditions in6×SSC at about 45° C., followed by one or more washes in 0.2×SSC, 0.1%SDS at 65° C.; and (4) very high stringency hybridization conditions are0.5M sodium phosphate, 7% SDS at 65° C., followed by one or more washesat 0.2×SSC, 1% SDS at 65° C. Very high stringency conditions (4) are thepreferred conditions and the ones that should be used unless otherwisespecified. The invention includes nucleic acids that hybridize with low,medium, high, or very high stringency to a nucleic acid described hereinor to a complement thereof, e.g., nucleic acids encoding a bindingprotein described herein. The nucleic acids can be the same length orwithin 30, 20, or 10% of the length of the reference nucleic acid. Thenucleic acid can correspond to a region encoding an immunoglobulinvariable domain sequence.

An integrin binding protein may have mutations relative to a bindingprotein described herein (e.g., a conservative or non-essential aminoacid substitutions), which do not have a substantial effect on theprotein functions. Whether or not a particular substitution will betolerated, i.e., will not adversely affect biological properties, suchas binding activity can be predicted, e.g., using the method of Bowie,et al. (1990) Science 247:1306-1310.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). It is possiblefor many framework and CDR amino acid residues to include one or moreconservative substitutions.

Consensus sequences for biopolymers can include positions which can bevaried among various amino acids. For example, the symbol “X” in such acontext generally refers to any amino acid (e.g., any of the twentynatural amino acids or any of the nineteen non-cysteine amino acids).Other allowed amino acids can also be indicated for example, usingparentheses and slashes. For example, “(A/W/F/N/Q)” means that alanine,tryptophan, phenylalanine, asparagine, and glutamine are allowed at thatparticular position.

A “non-essential” amino acid residue is a residue that can be alteredfrom the wild-type sequence of the binding agent, e.g., the antibody,without abolishing or more preferably, without substantially altering abiological activity, whereas an “essential” amino acid residue resultsin such a change.

The terms “polypeptide” or “peptide” (which may be used interchangeably)refer to a polymer of three or more amino acids linked by a peptidebond, e.g., between 3 and 30, 12 and 60, or 30 and 300, or over 300amino acids in length. The polypeptide may include one or more unnaturalamino acids. Typically, the polypeptide includes only natural aminoacids. A “protein” can include one or more polypeptide chains.Accordingly, the term “protein” encompasses polypeptides. A protein orpolypeptide can also include one or more modifications, e.g., aglycosylation, amidation, phosphorylation, and so forth. The term “smallpeptide” can be used to describe a polypeptide that is between 3 and 30amino acids in length, e.g., between 8 and 24 amino acids in length.

The term “cognate ligand” refers to a naturally occurring ligand of anintegrin, including naturally occurring variants thereof (e.g., splicevariants, naturally occurring mutants, and isoforms).

The term “mimic,” in the context of a mimic of a conformation of anintegrin or portion thereof, refers to a modified integrin which has abias for at least one particular conformation relative to a naturallyoccurring integrin, or portion thereof.

Statistical significance can be determined by any art known method.Exemplary statistical tests include: the Students T-test, Mann Whitney Unon-parametric test, and Wilcoxon non-parametric statistical test. Somestatistically significant relationships have a P value of less than 0.05or 0.02. Particular binding proteins may show a difference, e.g., inspecificity or binding, that are statistically significant (e.g., Pvalue <0.05 or 0.02). The terms “induce”, “inhibit”, “potentiate”,“elevate”, “increase”, “decrease” or the like, e.g., which denotedistinguishable qualitative or quantitative differences between twostates, and may refer to a difference, e.g., a statistically significantdifference, between the two states.

Other features and advantages of the instant invention will become moreapparent from the following detailed description and claims. Embodimentsof the invention can include any combination of features describedherein. In no case does the term “embodiment” operate to exclude one ormore other features disclosed herein, e.g., in another embodiment. Thecontents of all references, patent applications (published andunpublished) and published patents, cited throughout this applicationare hereby expressly incorporated by reference. This application alsoincorporates by reference the 2003 Food and Drug Administration(FDA)-approved product label for RAPTIVA®.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a graph from an exemplary experiment that evaluates DX-1999(also known as D2-57) binding to HA cells (cells expressing an LFA-1with an I-domain locked in the high affinity conformation) relative toLA cells (cells expressing an LFA-1 with an I-domain locked in the lowaffinity conformation).

DETAILED DESCRIPTION

This disclosure provides, inter alia, binding proteins (e.g.,antibodies) that bind to an integrin in an activated conformation, e.g.,activated LFA-1 (“aLFA-1”), e.g., relative to a non-activatedconformation of LFA-1. In one embodiment, the binding proteins inhibitat least one function of an aLFA-1, e.g., inhibit a binding interactionbetween aLFA-1 and a cognate ligand of aLFA-1, e.g., an ICAM protein.The binding proteins can be used to treat or prevent an inflammatorydisorder or other disorder described herein.

LFA1

Lymphocyte function-associated antigen-1 (LFA-1) is a member of theleukocyte integrin subfamily. LFA-1 is a heterodimer of an integrinalpha subunit, αL (CD11a), and a beta subunit β2 (CD18).

Other integrins of the leukocyte integrin subfamily also include the β2subunit (CD18), but have distinct alpha subunits. For example, MAC-1 isa heterodimer of β2 and αM (CD11b). p150.95 is a heterodimer of β2 andαX (CD11c). Springer, T A (1990) Nature 346:425-433; Larson, R S andSpringer T A, (1990) Immunol Rev 114:181-217; Van der Vieren, M et al.(1995) Immunity 3:683-690. The leukocyte integrins mediate a wide rangeof adhesive interactions that are essential for normal immune andinflammatory responses. LFA-1 binding to its cognate ligand can resultin conformational changes to LFA-1 with significant cellular effects.LFA-1 functions that are mediated by clustering alone appear to besecondary to those mediated by ligand binding. See, e.g., Kim et al.(2004) J. Cell Biol. 167:1241.

An exemplary amino acid sequence of the αsubunit of human LFA-1 (αL orCD11a) is as follows (gi|4504757|ref|NP_(—)002200.1|):

(SEQ ID NO: 30) MKDSCITVMAMALLSGFFFFAPASSYNLDVRGARSFSPPRAGRHFGYRVLQVGNGVIVGAPGEGNSTGSLYQCQSGTGHCLPVTLRGSNYTSKYLGMTLATDPTDGSILACDPGLSRTCDQNTYLSGLCYLFRQNLQGPMLQGRPGFQECIKGNVDLVFLFDGSMSLQPDEFQKILDFMKDVMKKLSNTSYQFAAVQFSTSYKTEFDFSDYVKWKDPDALLKHVKHMLLLTNTFGAINYVATEVFREELGARPDATKVLIIITDGEATDSGNIDAAKDIIRYIIGIGKHFQTKESQETLHKFASKPASEFVKILDTFEKLKDLFTELQKKIYVIEGTSKQDLTSFNMELSSSGISADLSRGHAVVGAVGAKDWAGGFLDLKADLQDDTFIGNEPLTPEVRAGYLGYTVTWLPSRQKTSLLASGAPRYQHMGRVLLFQEPQGGGHWSQVQTIHGTQISGYFGGELCGVDVDQDGETELLLIGAPLFYGEQRGGRVFIYQRRQLGFEEVSELQGDPGYPLGRFGEAITALTDINGDGLVDVAVGAPLEEQGAVYIFNGRHGGLSPQPSQRIEGTQVLSGIQWFGRSIHGVKDLEDGGLADVAVGAESQMIVLSSRPVVDMVTLMSFSPAEIPVHEVECSYSTSNKMKEGVNITICFQIKSLYPQFQGRLVANLTYTLQLDGHRTRRRGLFPGGRHELRRNIAVTTSMSCTDFSFHFPVCVQDLISPINVSLNFSLWEEEGTPRDQRAQGKDIPPILRPSLHSETWEIPFEKNCGEDKKCEANLRVSFSPARSRALRLTAFASLSVELSLSNLEEDAYWVQLDLHFPPGLSFRKVEMLKPHSQIPVSCEELPEESRLLSRALSCNVSSPIFKAGSHVALQMMFNTLVNSSWGDSVELHANVTCNNEDSDLLEDNSATTIIPILYPIINILIQDQEDSTLYVSFTPKGPKIHQVKHMYQVRIQPSIHDHNIPTLEAVVGVPQPPSEGPITHQWSVQMEPPVPCHYEDLERLPDAAEPCLPGALFRCPVVFRQEILVQVIGTLELVGEIEASSMFSLCSSLSISFNSSKHFHLYGSNASLAQVVMKVDVVYEKQMLYLYVLSGIGGLLLLLLIFIVLYKVGFFKRNLKEKMEAGRGVPNGIPAEDSEQLASGQEAGDPGCLKPLHEKDSESGGGKD.

An exemplary amino acid sequence of the β subunit of human LFA-1 (β2) isas follows (gi|4557886|ref|NP_(—)000202.1):

(SEQ ID NO: 31) MLGLRPPLLALVGLLSLGCVLSQECTKFKVSSCRECIESGPGCTWCQKLNFTGPGDPDSIRCDTRPQLLMRGCAADDIMDPTSLAETQEDHNGGQKQLSPQKVTLYLRPGQAAAFNVTFRRAKGYPIDLYYLMDLSYSMLDDLRNVKKLGGDLLRALNEITESGRIGFGSFVDKTVLPFVNTHPDKLRNPCPNKEKECQPPFAFRHVLKLTNNSNQFQTEVGKQLISGNLDAPEGGLDAMMQVAACPEEIGWRNVTRLLVFATDDGFHFAGDGKLGAILTPNDGRCHLEDNLYKRSNEFDYPSVGQLAHKLAENNIQPIFAVTSRMVKTYEKLTEIIPKSAVGELSEDSSNVVHLIKNAYNKLSSRVFLDHNALPDTLKVTYDSFCSNGVTHRNQPRGDCDGVQINVPITFQVKVTATECIQEQSFVIRALGFTDIVTVQVLPQCECRCRDQSRDRSLCHGKGFLECGICRCDTGYIGKNCECQTQGRSSQELEGSCRKDNNSIICSGLGDCVCGQCLCHTSDVPGKLIYGQYCECDTINCERYNGQVCGGPGRGLCFCGKCRCHPGFEGSACQCERTTEGCLNPRRVECSGRGRCRCNVCECHSGYQLPLCQECPGCPSPCGKYISCAECLKFEKGPFGKNCSAACPGLQLSNNPVKGRTCKERDSEGCWVAYTLEQQDGMDRYLIYVDESRECVAGPNIAAIVGGTVAGIVLIGILLLVIWKALIHLSDLREYRRFEKEKLKSQWNNDNP LFKSATTTVMNPKFAES.

Proteins that preferentially bind to an activated leukocyte integrin canbe used to modulate a leukocyte activity and a physiological activitymediated by a leukocyte, e.g., an activated leukocyte. Such bindingproteins can be used to modulate (e.g., inhibit) leukocyte migration,leukocyte adherence, or inflammation.

Integrins can adopt a plurality of conformations, including an activatedand a non-activated conformation. Additional conformationalintermediates are also available. The conformation of an integrin can bebiased, for example, by modifying the amino acid sequence of theintegrin. A bias in conformation can be introduce thin a single domainof an integrin, e.g., within an integrin I-domain, a β-propeller domain,or between domains, or between subunits. In one embodiment, the integrinis modified by the engineering of an intra-molecular or inter-moleculardisulfide bond. Modified integrin molecules can be used as mimics of aconformation of a naturally occurring integrin.

The N-terminal region of the integrin α subunits contains seven repeatsof about 60 amino acids each, and has been predicted to fold into a7-bladed β-propeller domain (Springer, T A (1997) Proc Natl Acad Sci USA94:65-72). The leukocyte integrin α subunits (such as the α1, α2, α10,α11, αL, αM, αD, αX, and αE subunits) contain an inserted domain orI-domain of about 200 amino acids (Larson, R S et al. (1989) J Cell Biol108:703-712; Takada, Yet al. (1989) EMBO J 8:1361-1368; Briesewitz, R etal. (1993) J Biol Chem 268:2989-2996; Shaw, S K et al. (1994) J BiolChem 269:6016-6025; Camper, L et al. (1998) J Biol Chem273:20383-20389). The I-domain is predicted to be inserted betweenβ-sheets 2 and 3 of the β-propeller domain. The three dimensionalstructure of the αM, αL, α1 and α2 I-domains has been solved and showsthat it adopts the dinucleotide-binding fold with a unique divalentcation coordination site designated the metal ion-dependent adhesionsite (MIDAS) (Lee, J-O, et al. (1995) Structure 3:1333-1340; Lee, J-O,et al. (199S) Cell 80:631-638; Qu, A and Leahy, D J (1995) Proc NatlAcad Sci USA 92:10277-10281; Qu, A and Leahy, D J (1996) Structure4:931-942; Emsley, J et al. (1997) J Biol Chem 272:28512-28517; Baldwin,E T et al. (1998) Structure 6:923-935; Kallen, J et al. (1999) J MolBiol 292:1-9). The C-terminal region of the αM subunit has beenpredicted to fold into a β-sandwich structure (Lu, C et al. (1998) JBiol Chem 273:15138-15147).

US 2002-0123614 describes, inter alia, exemplary methods for obtainingand using conformationally biased integrin molecules. In one embodiment,an integrin is locked in a particular conformation using a disulfidebond. Computational algorithms for designing and/or modeling proteinconformations are described, for example, in WO 98/47089. The SSBONDprogram (Hazes, B and Dijkstra, B W (1988) Protein Engineering2:119-125) can be used to identify positions where disulfide bonds canbe introduced in a protein structure by mutating appropriatelypositioned pairs of residues to cysteine.

Disulfide bond formation can occur between two cysteine residues thatare appropriately positioned within the three-dimensional structure of aprotein. Accordingly, a protein can be stabilized in a desiredconformation by introducing at least one cysteine substitution into theamino acid sequence such that a disulfide bond is formed. Theintroduction of a single cysteine substitution is performed incircumstances in which an additional cysteine residue is present in thenative amino acid sequence of the protein at an appropriate positionsuch that a disulfide bond is formed. More commonly, two cysteinesubstitutions are introduced into the amino acid sequence of the proteinat positions that allow a disulfide bond to form, thereby stabilizingthe protein in a desired conformation. In another embodiment, thedistance between the Cβ carbons of the residues that are substituted forcysteine is 3.00-8.09 Å. In yet another embodiment, the distance betweenthe Cβ carbons in the disulfide bond is in the range of 3.41-7.08 Å.

Typically, cysteine substitutions are introduced such that the formationof a disulfide bond is favored only in one protein conformation, suchthat the protein is stabilized in that particular conformation. Cysteinesubstitutions can be produced by mutagenesis of DNA encoding thepolypeptides of interest (e.g., integrin polypeptides). For example, anisolated nucleic acid molecule encoding a modified integrin I-domainpolypeptide can be created by introducing one or more nucleotidesubstitutions into the nucleotide sequence of an integrin gene such thatone or more codons, e.g., cysteine codons, are introduced into theencoded protein. Mutations can be introduced into a nucleic acidsequence by standard techniques, such as site-directed mutagenesis andPCR-mediated mutagenesis.

Additional methods for obtaining and using integrins in a lockedconformation are described, e.g., in Shimaoka, M et al. (2003) Cell112,99-111; Shimaoka, M et al. (2002) Annu. Rev. Biophys. Biomol.Struct. 31, 485-516; and Shimaoka, M et al. (2001) Proc Natl Acad SciUSA 98:6009-6014. Luo et al. (2003) Proc Natl Acad Sci USA.100(5):2403-8 describe a conformationally biased integrin in which aglycan moiety is used to alter conformational preference. Luo et al. JBiol. Chem. 2003 Dec. 16 (Epub ahead of print), PMID: 14681220, describeadditional conformationally biased, e.g., disulfide lockedconformations.

For example, a conformationally biased integrin can include a modifiedintegrin I-domain that is biased towards (e.g., locked in) the openconformation or a closed conformation. The open conformation may bind toa cognate ligand of the integrin with high affinity.

A disulfide locked molecule can be produced from a nucleic acid sequencethat includes at least one codon substitution that inserts one or more(e.g., two) cysteine codons. The codons can be positions such that inthe encoded protein, the distance between the Cβ carbons of the residuesthat are substituted for cysteines is in the range of 3.00-8.09Angstroms e.g., as predicted by protein modeling. In a furtherembodiment, the distance between the Cβ carbons in the disulfide bond isin the range of 3.41-7.08 Angstroms.

Examples of integrin I-domains that are conformationally biased towardsa particular conformation, e.g., an active “open” conformation, or anon-activated “closed” conformation include the following. The αLK287C/K294C, E284C/E301C, L161C/F299C, K160C/F299C, L161C/T300C, andL289C/K294C mutants, and the αM Q163C/Q309C and D294C/Q31 IC. mutantsare stabilized in “open” conformations that bind the cognate ligand withhigh or intermediate affinity, whereas the αL L289C/K294C mutant and theαM Q163C/R313c mutants are stabilized in non-activated “closed”conformations that do not bind to the cognate ligand. The affinity ofE284C/E301C for the cognate ligand is nearly comparable to that ofK287C/K294C, e.g., high-affinity. The affinity of L161C/F299C,K160C/F299C, and L161C/T300C for the cognate ligand are significantlyhigher than wild-type, but 20-30 times lower than high-affinity αLI-domain, K287C/K294C. L161C/F299C, K160C/F299C, and L161C/T300C arereferred to herein as intermediate affinity αL I-domains.

The I-domain of αL is described as follows, with secondary structureinformation below:

(SEQ ID NO: 32) 1 GNVDLVFLF DGSMSLQPDE FQKILDFMKD VMKKLSNTSY QFAAVQFSTS    EEEEEEE E BTTS HHH HHHHHHHHHH HHHHTTTSSE EEEEEEESSS 50YKTEFDFSDY VKRKDPDALL KHVKHMLLLT NTFGAINYVA TEVFREELGAEEESB HHHH HHHTTHHHHT SS     B    HHHHHHHHH HHTTTGGGT 100RPDATKVLII ITDGEATDSG NIDAAKDIIR YIIGIGKHFQ TKESQETLHK TTSEEEEEE EE S  S      GGGTTSEE EEEE SS    STTTGGGGTT 150FASKPASEFV KILDTFEKLK DLFTELQKKI TS SSHHHHE EETTTTTTTT TTT

See, e.g., PDB™ structures: (1) 1MQA “Crystal Structure Of High AffinityAlpha-1 I Domain In The Absence Of Ligand Or Metal” (mmdbId:21776); (2)1MQ9 “Crystal Structure Of High Affinity Alpha-1 I Domain With LigandMimetic Crystal Contact” (mmdbId:21775); (3) 1MQ8 “Crystal Structure OfAlpha-1 I Domain In Complex With Icam-1” (mmdbId:21774); and (4) 1MJN“Crystal Structure Of The Intermediate Affinity Al I Domain Mutant”(mmdbId:21755).

Conformationally biased integrin molecules may include just a modifiedintegrin I-domain from an integrin α subunit, or the entire mature αsubunit extracellular domain, or the entire mature α subunit, and/or maybe further associated with an integrin β subunit extracellular domainand/or entire subunit. In one embodiment, a modified integrin I-domainpolypeptide is a soluble protein, e.g., a heterodimeric soluble protein,or a monomeric soluble protein.

A model of the I-like domain of the integrin β-subunit that is supportedby experimental data (Huang, C et al. (2000) J Biol Chem 275:21514-24)has also been made. The data confirm the location of the key C-terminalα-helix that undergoes the dramatic 10 Angstrom conformational movementin I-domains. The I and I-like domains align well in this region.

Identification of aLFA-1 Binding Proteins

A number of methods can be used to identify proteins that bind to aLFA-1and other active integrins. Many of these methods useconformationally-biased integrin proteins as targets.

One exemplary method for identifying antibodies that bind to aLFA-1includes immunizing a non-human animal with a conformationally biasedLFA-1 protein or a conformationally biased domain thereof. Spleen cellscan be isolated from the immunized animal and used to produce hybridomacells using standard methods. In one embodiment, the non-human animalincludes one or more human immunoglobulin genes.

Another exemplary method for identifying proteins that bind to aLFA-1includes: providing a library of proteins and selecting from the libraryone or more proteins that bind to a conformationally biased molecule,e.g., a conformationally biased integrin, e.g., aLFA-1. The selectioncan be performed in a number of ways. For example, the library can beprovided in the format of a display library or a protein array. Prior toselecting, the library can be pre-screened (e.g., depleted) to removemembers that interact with a non-target molecule, e.g., an LFA-1molecule in the non-activated conformation.

The conformationally biased target molecule can be tagged andrecombinantly expressed. In one embodiment, the conformationally biasedtarget molecule is purified and attached to a support, e.g., to affinitybeads, or paramagnetic beads or other magnetically responsive particles.

A conformationally biased target molecule can also be expressed on thesurface of a cell. Members of the display library that specifically bindto the cell can be selected. It is also possible to use an endogenous orother wild-type form of an integrin. For example, members of the displaylibrary that specifically bind to a cell, only if the integrin isactivated, can be selected.

Expression Libraries

In one embodiment, a display library or other expression library is usedto identify proteins that bind to an integrin in an activatedconformation, e.g., aLFA-1. A display library is a collection ofentities; each entity includes an accessible protein component (e.g., aFab or scFv) and a recoverable component (e.g., a nucleic acid) thatencodes or identifies the protein component. The protein component canbe of any length, e.g. from three amino acids to over 300 amino acids.In a selection, the protein component of each member of the library isprobed with a conformationally biased integrin protein and if theprotein component binds to the protein, the display library member isidentified, e.g., by retention on a support. The protein component caninclude one or more immunoglobulin variable domains or variants ofanother domain. Methods for making libraries of immunoglobulin domainsare well known. See, e.g., U.S. Application Ser. No. 60/546,354, filedon Feb. 19, 2004, US 2004-0005709, and US 2002-0102613.

Retained display library members are recovered from the support andanalyzed. The analysis can include amplification and a subsequentselection under similar or dissimilar conditions. For example, positiveand negative selections can be alternated. The analysis can also includedetermining the amino acid sequence of the protein component andpurification of the protein component for detailed characterization.

A variety of formats can be used for display libraries. Examples includethe following.

Phage Display. One format utilizes viruses, particularly bacteriophages.This format is termed “phage display.” The protein component istypically covalently linked to a bacteriophage coat protein. The linkageresults from translation of a nucleic acid encoding the proteincomponent fused to the coat protein. The linkage can include a flexiblepeptide linker, a protease site, or an amino acid incorporated as aresult of suppression of a stop codon. Phage display is described, forexample, in U.S. Pat. No. 5,223,409; Smith (1985) Science 228:1315-1317;WO 92/18619; WO 91/17271; WO 92/20791; WO 92/15679; WO 93/01288; WO92/01047; WO 92/09690; WO 90/02809; de Haard et al. (1999) J. Biol.Chem. 274:18218-30; Hoogenboom et al. (1998) Immunotechnology 4:1-20;and Hoogenboom et al. (2000) Immunol Today 2:371-8.

Phage display systems have been developed for filamentous phage (phagef1, fd, and M13) as well as other bacteriophage. The filamentous phagedisplay systems typically use fusions to a minor coat protein, such asgene III, to present the protein component on the surface of thebacteriophage. It is also possible to physically associate the proteinbeing displayed to the coat using a non-peptide linkage.

Bacteriophage displaying the protein component can be grown andharvested using standard phage preparatory methods, e.g. PEGprecipitation from growth media. After selection of individual displayphages, the nucleic acid encoding the selected protein components can beisolated from cells infected with the selected phages or from the phagethemselves, after amplification. Individual colonies or plaques can bepicked, the nucleic acid isolated and sequenced.

Other Display Formats. Other display formats include cell based display(see, e.g., WO 03/029456), protein-nucleic acid fusions (see, e.g., U.S.Pat. No. 6,207,446), and ribosome display (See, e.g., Mattheakis et al.(1994) Proc. Natl. Acad. Sci. USA 91:9022 and Hanes et al. (2000) Nat.Biotechnol. 18:1287-92; Hanes et al. (2000) Methods Enzymol. 328:404-30;and Schaffitzel et al. (1999) J Immunol Methods. 231(1-2):119-35).

Epitope Specific Binding proteins. Display technology can also be usedto obtain binding proteins, e.g., antibodies, that bind to particularepitopes of a target. Epitopes can be classified as “conformational” or“sequential”. Conformational epitopes involve amino-acid residues thathave a defined relative orientation in a properly folded target eventhough the amino acids may be substantially separated in the sequence(e.g., separated by at least one, two, four, six, eight or ten aminoacids). Sequential epitopes involve short portions of the polypeptidechain that bind an antibody whatever the folding state of the protein(e.g., native or unfolded). Binding proteins for conformational epitopescan be identified, for example, by using competing non-target moleculesthat lack the particular epitope or are mutated within the epitope,e.g., with alanine. Such non-target molecules can be used in a negativeselection procedure as described below, as competing molecules whenbinding a display library to the target, or as a pre-elution agent,e.g., to capture in a wash solution dissociating display library membersthat are not specific to the target. In another implementation, epitopespecific binding proteins are identified by eluting display librarymembers with a competing binding protein that binds to the epitope ofinterest on the target molecule. Binding proteins that bind sequentialepitopes can be selected, for example, using short peptides that haveamino-acid sequences found in a target protein. Often binding proteinsthat bind to conformational epitopes also bind weakly to one or anotherpeptide that contains some of the amino acids involved in theconformational epitope. Thus, one can select for binding to a peptide atvery low stringency and then select for binding to the folded targetprotein.

Affinity Maturation. In one embodiment, a binding protein that binds toa target is modified, e.g., by mutagenesis, to provide a pool ofmodified binding proteins. The modified binding proteins are thenevaluated to identify one or more altered binding proteins which havealtered functional properties (e.g., improved binding, improvedstability, lengthened stability in vivo). In one implementation, displaylibrary technology is used to select or screen the pool of modifiedbinding proteins. Higher affinity binding proteins are then identifiedfrom the second library, e.g., by using higher stringency or morecompetitive binding and washing conditions. Other screening techniquescan also be used.

In some implementations, the mutagenesis is targeted to regions known orlikely to be at the binding interface. If, for example, the identifiedbinding proteins are antibodies, then mutagenesis can be directed to theCDR regions of the heavy or light chains as described herein. Further,mutagenesis can be directed to framework regions near or adjacent to theCDRs, e.g., framework regions, particular within ten, five, or threeamino acids of a CDR junction. In the case of antibodies, mutagenesiscan also be limited to one or a few of the CDRs, e.g., to make step-wiseimprovements.

In one embodiment, mutagenesis is used to make an antibody more similarto one or more germline sequences. One exemplary germlining method caninclude: identifying one or more germline sequences that are similar(e.g., most similar in a particular database) to the sequence of theisolated antibody. Then mutations (at the amino acid level) can be madein the isolated antibody, either incrementally, in combination, or both.For example, a nucleic acid library that includes sequences encodingsome or all possible germline mutations is made. The mutated antibodiesare then evaluated, e.g., to identify an antibody that has one or moreadditional germline residues relative to the isolated antibody and thatis still useful (e.g., has a functional activity). In one embodiment, asmany germline residues are introduced into an isolated antibody aspossible.

In one embodiment, mutagenesis is used to substitute or insert one ormore germline residues into a CDR region. For example, the germline CDRresidue can be from a germline sequence that is similar (e.g., mostsimilar) to the variable region being modified. After mutagenesis,activity (e.g., binding or other functional activity) of the antibodycan be evaluated to determine if the germline residue or residues aretolerated. Similar mutagenesis can be performed in the frameworkregions.

Selecting a germline sequence can be performed in different ways. Forexample, a germline sequence can be selected if it meets a predeterminedcriteria for selectivity or similarity, e.g., at least a certainpercentage identity, e.g., at least 75, 80, 85, 90, 91, 92, 93, 94, 95,96, 97, 98, 99, or 99.5% identity. The selection can be performed usingat least 2, 3, 5, or 10 germline sequences. In the case of CDR1 andCDR2, identifying a similar germline sequence can include selecting onesuch sequence. In the case of CDR3, identifying a similar germlinesequence can include selecting one such sequence, but may includingusing two germline sequences that separately contribute to theamino-terminal portion and the carboxy-terminal portion. In otherimplementations more than one or two germline sequences are used, e.g.,to form a consensus sequence.

In one embodiment, with respect to a particular reference variabledomain sequence, e.g., a sequence described herein, a related variabledomain sequence has at least 30, 40, 50, 60, 70, 80, 90, 95 or 100% ofthe CDR amino acid positions that are not identical to residues in thereference CDR sequences, residues that are identical to residues atcorresponding positions in a human germline sequence (i.e., an aminoacid sequence encoded by a human germline nucleic acid).

In one embodiment, with respect to a particular reference variabledomain sequence, e.g., a sequence described herein, a related variabledomain sequence has at least 30, 50, 60, 70, 80, 90 or 100% of the FRregions are identical to FR sequence from a human germline sequence,e.g., a germline sequence related to the reference variable domainsequence.

Accordingly, it is possible to isolate an antibody which has similaractivity to a given antibody of interest, but is more similar to one ormore germline sequences, particularly one or more human germlinesequences. For example, an antibody can be at least 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 99.5% identical to a germline sequence in a regionoutside the CDRs (e.g., framework regions). Further an antibody caninclude at least 1, 2, 3, 4, or 5 germline residues in a CDR region, thegermline residue being from a germline sequence of similar (e.g., mostsimilar) to the variable region being modified. Germline sequences ofprimary interest are human germline sequences. The activity of theantibody (e.g., the binding activity) can be within a factor or 100, 10,5, 2, 0.5, 0.1, and 0.001 of the original antibody. An exemplarygermline sequences include VKI-O2, VL2-1, VKIII-L2::JK2, vg3-23,V3-23::JH4, and V3-23::JK6.

Some exemplary mutagenesis techniques include: error-prone PCR (Leung etal. (1989) Technique 1:11-15), recombination (see, e.g., U.S. Ser. No.10/279,633), DNA shuffling using random cleavage (Stemmer (1994) Nature389-391; termed “nucleic acid shuffling”), RACHITT™ (Coco et al. (2001)Nature Biotech. 19:354), site-directed mutagenesis (Zoller et al. (1987)Nucl Acids Res 10:6487-6504), cassette mutagenesis (Reidhaar-Olson(1991) Methods Enzymol. 208:564-586) and incorporation of degenerateoligonucleotides (Griffiths et al. (1994) EMBO J. 13:3245).

In one example of affinity maturation the methods described herein areused to first identify a binding protein from a display library thatbinds an aLFA-1 with at least a minimal binding specificity for a targetor a minimal activity, e.g., an equilibrium dissociation constant forbinding of less than 1 nM, 10 nM, or 100 nM. The nucleic acid sequenceencoding the initial identified binding protein are used as a templatenucleic acid for the introduction of variations, e.g., to identify asecond binding protein that has enhanced properties (e.g., bindingaffinity, kinetics, or stability) relative to the initial bindingprotein. Alternatively, the amino-acid sequence of one or more CDRs canbe used as a guide for design of a nucleic acid library that includesnucleic acids encoding the isolated sequence and many neighboringsequences. Such diversified nucleic acids can be introduced into adisplay vector containing the initial isolate and improved variants areselected from the library.

Off-Rate Selection. Since a slow dissociation rate can be predictive ofhigh affinity, particularly with respect to interactions betweenpolypeptides and their targets, the methods described herein can be usedto isolate binding proteins with a desired kinetic dissociation rate(i.e. reduced) for a binding interaction to a target.

To select for slow dissociating binding proteins from a display library,the library is contacted to an immobilized target. The immobilizedtarget is then washed with a first solution that removesnon-specifically or weakly bound biomolecules. Then the immobilizedtarget is eluted with a second solution that includes a saturationamount of free target, i.e., replicates of the target that are notattached to the particle. The free target binds to biomolecules thatdissociate from the target. Rebinding is effectively prevented by thesaturating amount of free target relative to the much lowerconcentration of immobilized target.

The second solution can have solution conditions that are substantiallyphysiological or that are stringent. Typically, the solution conditionsof the second solution are identical to the solution conditions of thefirst solution. Fractions of the second solution are collected intemporal order to distinguish early from late fractions. Later fractionsinclude biomolecules that dissociate at a slower rate from the targetthan biomolecules in the early fractions.

Further, it is also possible to recover display library members thatremain bound to the target even after extended incubation. These caneither be dissociated using chaotropic conditions or can be amplifiedwhile attached to the target. For example, phage bound to the target canbe contacted to bacterial cells.

Selecting and Screening for Specificity. “Selection”, in the context ofa display library, refers to a process in which many members of adisplay library are allowed to contact the target and those that bindare recovered and propagated. The selection can be from a library havingnumerous members, e.g., more than 10¹⁰ members. “Screening”, in thecontext of a display library, refers to a process in which isolatedmembers of the library are tested singly for binding to the target.Through automation, thousands of candidates may be screened in a highlyparallel process. The display library selection methods described hereincan include a selection process that discards display library membersthat bind to a non-target molecule.

Examples of non-target molecules, e.g., for an LFA-1 binding antibody,include, e.g., integrins other than LFA-1. In another example, for anaLFA-1 binding antibody, e.g., an antibody that preferentially binds toactivated LFA-1, the non-target molecule can be an LFA-1 molecule in aconformation other than activated, e.g., a non-activated conformation.

In one implementation, a so-called “negative selection” step is used todiscriminate between the target and related non-target molecule and arelated, but distinct non-target molecule. The display library or a poolthereof is contacted to the non-target molecule. Members of the samplethat do not bind the non-target are collected and used in subsequentselections for binding to the target molecule or even for subsequentnegative selections. The negative selection step can be prior to orafter selecting library members that bind to the target molecule.

In another implementation, a screening step is used. After displaylibrary members are isolated for binding to the target molecule, eachisolated library member is tested for its ability to bind to anon-target molecule (e.g., a non-target listed above). For example, ahigh-throughput ELISA screen can be used to obtain this data. The ELISAscreen can also be used to obtain quantitative data for binding of eachlibrary member to the target. The non-target and target binding data arecompared (e.g., using a computer and software) to identify librarymembers that specifically bind to aLFA-1.

The display library selection and screening methods described herein caninclude a selection or screening process that selects for displaylibrary members that bind to specific sites on the target molecule. Forexample, elution with high concentration of an antibody described hereinselects for phage that bind to the epitope bound by such an antibody.One can screen for a phage that binds to a particular epitope of aLFA-1by performing ELISAs with and without a competing antibody thatrecognizes the epitope in the buffer.

Secondary Screening Methods

Display libraries can be used to select candidate display librarymembers that bind to the target. Each such candidate library member orany candidate aLFA-1 binding protein can be further analyzed, e.g., tofurther characterize its binding properties for the target. Eachcandidate display library member can be subjected to one or moresecondary screening assays. The assay can be for a binding property, aphysiological property (e.g., cytotoxicity, renal clearance,immunogenicity), a structural property (e.g., stability, conformation,oligomerization state) or another functional property (e.g. ability tomodulate an activity of an integrin-expressing cell, e.g., a leukocyte,or ability to modulate inflammation or an inflammationassociated-response). The same assay can be used repeatedly, but withvarying conditions, e.g., to determine pH, ionic, or thermalsensitivities.

As appropriate, the assays can use the display library member directly,a recombinant polypeptide produced from the nucleic acid encoding adisplayed polypeptide, a synthetic peptide synthesized based on thesequence of a displayed polypeptide. In the case of a candidate aLFA-1binding protein from any source, the protein can be obtained, e.g., fromsuch a source or by recombinant production. Exemplary assays for bindingproperties include the following.

Exemplary Biological Assays

Candidate aLFA-1 binding proteins can be evaluated for their activity invitro (e.g., in a cell-free or cell-based system) or in vivo (e.g., inan animal model describe below). For example, the proteins can beevaluated for their ability to inhibit an activity of LFA-1 expressingcells, e.g., a binding activity of an LFA-1 expressing cell. In anotherexample, the proteins can be evaluated for their ability to target cellsthat present activated LFA-1.

The binding of LFA-1 expressing cells to a cognate ligand can beevaluated, e.g., using cellular assays. ICAM-1 is expressed, e.g., onleukocytes, endothelium, and dermal fibroblasts (Dustin et al., J.Immunol. 137: 245-254 (1986)), ICAM-2 expressed on resting endotheliumand lymphocytes (de Fougerolles et al., J. Exp. Med. 174: 253-267(1991)), and ICAM-3 expressed on monocytes and resting lymphocytes (deFougerolles et al., J. Exp. Med. 179: 619-629 (1994)). Accordingly, celladhesion assays (e.g., using fluorescently labeled cells) can beperformed between LFA-1 expressing cells and other leukocytes,endothelial cells, monocytes, and dermal fibroblasts.

Another exemplary assay for ICAM binding is as follows: ICAM-1 ispurified from human tonsil, and coated on 96-well plates as describedpreviously (Lu and Springer, (1997) J Immunol 159:268-278). LFA-1expressing cells are labeled with a florescence dye2′,7′-bis-(carboxyethyl)-5(and -6)-carboxyfluorescein, acetoxymethylester (BCECF-AM), and resuspended at about 1·10⁶/ml in L15/FBS. 50 μl ofcell suspension is mixed in ICAM-1 coated wells with an equal volume ofL15/FBS in the absence or presence of a test compound (e.g., a candidateaLFA-1 binding protein). The assays can be performed in the presence andabsence of an activating monoclonal antibody (CBRLFA-1/2, 10 μg/ml).

For testing the effect of divalent cations, BCECF-AM-labeled cells arewashed twice with TS buffer, pH7.5 (20 mM Tris, pH 7.5, 150 mM NaCl)containing 5 mM EDTA, followed by two washes with TS buffer, pH7.5.Cells were then resuspended to 5·10⁵/ml in the TS buffer, pH7.5supplemented with 1 mM MgCl₂ and other divalent cations and 2 mM EDTA.100 μl of the cell suspension is added to ICAM-1 coated wells. Afterincubation at 37° C. for 30 minutes, unbound cells are washed off on aMicroplate AUTOWASHER™ (Bio-Tek Instruments, Winooski, Vt.). Thefluorescence content of total input cells and the bound cells in eachwell is quantitated on a Fluorescent Concentration Analyzer (IDEXX,Westbrook, Me.). The number of bound cells can be expressed as apercentage of total input cells per sample well.

The following exemplary assay evaluates the effect of a test compound(e.g., an aLFA-1 binding protein) on the ability of test compound tomodulate cell-cell interactions that depend on LFA-1. The assay useslymphoma cell line EL-4 which expresses both murine LFA-1 and ICAM-1,and which exhibits LFA-1-dependent homotypic aggregation upon activationby PMA. Cells are incubated in a 96 well plate in the presence of 50ng/ml PMA and varying amounts of the test compound. After incubation for2 hours at 37° C., 5% CO₂, the degree of aggregation was scored underthe microscope as follows: 0 indicated that essentially no cells areclustered; 1 indicated that <10% of cells are aggregated; 2 indicatedclustering of <50%; 3 indicated that up to 100% of cells were in small,loose aggregates; 4 indicated that nearly 100% of cells are in largerclusters; and 5 indicated that nearly 100% of cells are in very large,tight clusters.

Still another exemplary assay evaluates the ability of a test compoundto inhibit LFA-1 function in vivo. The assay includes visualizingmicrocirculation in the peripheral lymph node (LN) with intravitalmicroscopy. Briefly, a small bolus (20-50 μl) of LN cell suspensionsfrom TGFβ mice are retrogradely injected through a femoral arterycatheter and visualized in the subiliac LN by fluorescentepi-illumination from a video-triggered xenon arc stroboscope. Afterrecording control TGFβ cell behavior in the absence of test compound,the mouse was pretreated by intra-arterial injection of the testcompound (e.g., at a desired concentration) 5 minutes before T^(GFP)cell injection. Scenes can be recorded on videotape and off-lineanalysis was done. The rolling fraction can be calculated as percentageof the number rolling cells relative to the total number of TGFβ cellsthat entered a venule. The sticking (firm adhesion) fraction can bedetermined as the percentage of T^(GFP) cells becoming firmly adherentfor >20 seconds in the number of T^(GFP) cells that rolled in a venule.Results can be semi-quantitatively scored as follows: −: 0%, .+−.: 0-5%,+: 5-20%, ++: 20-40%, +++: 40-60%, ++++: 60-80%, +++++: 80-100%.

The vascular endothelium is a substrate with whichmonocytes/granulocytes can interact during adherence, diapedesis, anddifferentiation. An in vitro assay for monocyte/granulocyte interactionwith the vessel wall consists of binding radiolabeled or fluoresceinmonocyte/granulocyte preparations to cultured vascular endothelium, asdescribed in Arnaout et al., J. Cell Physiol. 137:305 (1988). Mentzer etal., J. Cell Physiol. 125:285 (1986) describes a lymphocyte adhesionassay. A granulocyte aggregation assay can be performed as described byArnaout et al., New Engl. J. Med. 306:693 (1982). Aggregation can beinduced by zymosan-activated autologous serum or with chemotacticpeptides, e.g. FMLP. Aggregation can then be recorded as incrementalchange in light transmission using a platelet aggregometer. The resultscan be confirmed by phase microscopy. Chemotaxis can be evaluated, e.g.,as described in Dana et al., J. Immunol. 137:3259 (1986).

A protein (e.g., an antibody described herein) can also be evaluated inculture for ability to modulate inflammation or an inflammatorydisorder. For example, cell culture is used to monitor adhesion ofleukocytes. A compound can be immobilized on a solid surface andadhesion of cells expressing an adhesion molecule can be evaluated forinteraction with the surface. Cells suitable for this assay include anyleukocytes, such as T cells, B cells, monocytes, eosinophils, andbasophils. Exemplary leukocyte cell lines include Jurkat and U937 cells.

In one embodiment, a protein (e.g., an antibody described herein) has astatistically significant effect in an assay described herein. An assayfor a protein can be compared to corresponding control assay, asappropriate, e.g., an assay lacking one or more components, e.g.,lacking the test compound, a particular cell, a particular antibody,cation, etc.

Animal Models

An aLFA-1 binding protein can be evaluated than animal model, e.g., ananimal model for an inflammatory disorder, a disorder characterized byexcessive LFA-1 activity, or a LFA-1 mediated disorder.

A number of animal models for psoriasis are available. The efficacy ofan integrin binding protein (e.g., an aLFA-1 binding antibody describedherein) can be tested in an animal model of psoriasis, e.g., in a BNXtransplanted psoriasis skin model, for example, the model described inWrone-Smith et al. (1996) J Clin Invest. 98(8):1878-87. Additionalexamples include the following. Schon et al. (1997) Nat. Med. 3:183-8describe a mouse having a murine psoriasis-like disorder. The mouse wascreated by reconstituting scid/scid mice with naive CD4⁺ T cells. Othermouse models for psoriasis have also utilized immunodeficient animals.Sugai et al. (1998) J Dermatol Sci 17:85-92 transplanted human psoriaticlesions onto scid mice. Yamamoto et al. (1998) J Dermatol Sci 17:8-14describe injecting staphylococcal enterotoxin B-stimulated lymphocytessubcutaneously under full-thickness psoriatic skin grafted onto severecombined immunodeficient (scid) mice. Sundberg et al. (1997)Pathobiology 65(5):271-86 describe the development and progression ofpsoriasiform dermatitis and systemic lesions in the flaky skin (fsn)mouse mutant. Flaky skin (fsn) mutant mice have been described as amouse model for psoriasis accompanied by hematological abnormalities.Hong et al. (1999) J. Immunol. 162:7480-7491 describe additional animalmodels of psoriasis. U.S. Pat. No. 6,410,824 describes producing ananimal model by transferring naive, immuno-competent T lymphocytes intoan immunodeficient animal host, along with at least one pro-inflammatorycytokine and a polyclonal activating agent. The engrafted T cells aretolerant to the major histocompatibility antigens of the host animal,but are mismatched at one or more minor histocompatibility loci. Theengrafted animals develop a chronic skin disorder that includeshistological features observed in human psoriasis, e.g. rete pegs,severe acanthosis and infiltration of Th1 cells into the dermis.

U.S. Pat. No. 6,462,020 describes an exemplary mouse model forarthritis, the induced Type H Collagen Arthritis Mouse Model. The mousemodel can be used to evaluate the effect of aLFA-1 binding proteins onthe histological, radiographic and clinical appearance of induced typeII collagen arthritis. The histopathology of arthritic lesions occurringin murine CIA share many similarities to that of rheumatoid arthritis(RA) in human patients. Murine CIA is a useful model to study potentialtherapeutic treatments of RA.

The following is an exemplary version of the murine CIA model. Materialsand Methods: Mice: DBA/1(2) male mice weighing 25 g (JacksonLaboratories, Bar Harbor, Me. or B&K Universal, Kent Wash.) are used forthis work. This strain of mouse is susceptible to CIA by the injectionof heterologous type II collagen. Bovine Collagen (BC), CompleteFreund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (ICFA) can beobtained from Sigma Chemical. Antigen for immunization is processed in0.1 M acetic acid and formulated with CFA or ICFA.

Induction of Arthritis. Immunization protocol: Mice are injected with100 μg of type II collagen in CFA at predetermined intervals during thestudy period.

The mice are examined at predetermined intervals for the development ofarthritis. Presumptive evidence of arthritis includes swelling anderythema of at least one toe joint on the front and/or rear feet on twoconsecutive observations.

Confirmatory Diagnosis of Arthritis. Histological Examination of joints:The toe joints of animals sacrificed at appropriate intervals areremoved, fixed, decalcified, embedded, in paraffin, sectioned, andstained for observation of general cellular and structural features andto detect cartilaginous matrix of the pannus of each joint, asappropriate. The degree of cellularity and areas of inflammation arequantified by using digitization of histological photomicrographs andapplying standard area and point counting techniques as described above.

Radiographic evaluation of toe joints is performed to detect theincidence of joint changes after immunization with type II collagen. Amammography imaging system has been modified for this work. The averagearea of soft tissue (pannus) of the joint is determined by analysis ofcomputer digitized radiographs, along with changes in density of theadjacent hard tissues by comparison with internal standards includedwith each radiograph. To serve as a baseline control for the changingdensity of the hard tissues and areas of panni, additional mice are usedover the same period and the density and area data compared. Thesignificance of the differences in density and area for control andexperimental mice is assessed using paired T-tests at each time point.

Arthritis Evaluation. Animals are observed daily for the onset ofarthritis. An arthritis index is derived by grading the severity ofinvolvement of each paw on a scale from 0 to 4. Scoring is based uponthe degree of peri-articular erythema and edema, as well as deformity ofthe joints. Swelling of hind paws is also quantitated by measuring thethickness of the ankle from the medial to the lateral malledus with aconstant tension caliper.

US 2003-0161810 provides a non-human animal model for an inflammatorydisorder (including rheumatoid arthritis). The animal described thereinincludes human synovial fluid. US 2003-0176389 describes a dextransodium sulfate-induced mouse model of colitis.

An aLFA-1 binding protein can be assayed for an effect on neutrophilmigration. One model for neutrophil migration is murine thioglycollateinduced peritonitis. Thioglycollate is injected i.p. to mice andimmediately thereafter the protein to be tested is given, e.g., by i.p.or s.c. The mice are killed after 4 hours, the peritoneal cavity lavagedand the total number of neutrophils in the lavage fluid is determined.

An aLFA-1 binding protein can be assayed for an effect onischemia/reperfusion injury. The protein can be tested, e.g., in a modelof heart ischemia/reperfusion injury (Abdeslam Oubenaissa et al.,Circulation, 94, Suppl. II, 254-258, 1996). The protein can also betested as follows:

Mice are treated with an aLFA-1 binding protein or a control. Miceweighing 20-25 g are anaesthetized with isoflurane and the right renalvessels are clamped using microvascular clamps for 60 min. After 60 minof ischemia, the microvascular clamps are removed. The left renalvessels (renal artery, vein and urethra) are ligated using a 4-0surgical suture. The left (non-ischemic) kidney is removed, and theabdominal cavity closed with 3-0 surgical suture. Control groups undergothe same procedures as the ischemia group, but without clamping of theright renal vessels.

Animals are sacrificed by CO₂ inhalation at 24 h, 1 week and 2 weeksfollowing reperfusion. Blood samples are collected by cardiac punctureinto a 3.0 ml VACUTAINER™ tube (Becton-Dickenson) containing 0.04 ml ofa 7.5% solution of K₃ EDTA immediately after sacrifice. Plasma isseparated and stored at −20° C. until further analysis. Plasma creatineand blood urea nitrogen (BUN) are analyzed. Following sacrifice, thekidney is flushed with physiological saline, immediately snap-frozen inliquid nitrogen and stored at −70° C. until analysis. Myeloperoxidaseactivity (MPO) in the kidney can be measured according to the method ofBradley et al (J. Invest. Dermatol., 78, 206-209, 1982).

An aLFA-1 binding protein can be assayed for an effect on vascularizedheterotopic heart transplantation. Recipient mice are treated with anaLFA-1 binding protein or a control. Mice donor hearts are implantedonto the recipients abdominal vessels: brachiocephalic trunk to aortaand right pulmonary artery to inferior vena cava with end-to-sideanastomoses using 11/0 Ethilon (Ethicon, Norderstedt, Germany)continuous sutures. Animals are closed in two layers with 6/0 Vicryl(Ethicon) and kept warm until fully recovered. Total ischemia times arein the range of 40-50 min of which 25-35 min are at 4° C. Duringanastomosis (10-15 min) the graft is kept cold.

After transplantation, graft function is monitored by daily assessmentof graft beat (palpation). Rejection is considered to be complete whenheart beat stops. In all experiments rejection is confirmed byhistological examination of the grafts.

An exemplary assay for reperfusion injury associated with myocardialinfarction in dogs is described, e.g. in Simpson et al., J. Clin.Invest. 81:624 (1988). Takeshima et al., Stroke, 23(2):247-252 (1992)describe a transient focal cerebral ischemia model in cats. Takeshima etal. used a microvascular clip to occlude the MCA and occluded CCAs bytightening previously placed ligatures. Lindsberg et al. J. Neurosurg.82:269-277 (1995) describe a rabbit model of severe spinal cord ischemia(by inflating the balloon of a catheter tip which had been introduced inthe abdominal aorta). Still additional models include the reversiblespinal cord model (involving a snare ligature occluding device) and anirreversible microsphere model. Clark et al., Stroke 22(7): 877-883(1991).

Bowes et al., Neurology 45:815-819 (1995) evaluated the ability ofparticular antibodies to enhance the efficacy of thrombolysis in arabbit cerebral embolism stroke model. In this model, numerous smallblood clots (formed by fragmenting a clot with a tissue homogenizer) areinjected into the rabbit's carotid circulation in order to achieveembolization. Neurologic function in each animal can be evaluated 18hours following embolization on a three point scale: (1) normalactivity; (2) abnormal activity; or (3) death. The amount of clotnecessary to produce permanent neurologic damage in 50% of the rabbits(ED.sub.50) is determined for each treatment group. Antibodies describedherein can be evaluated using this or a similar model to evaluateefficacy of thrombolysis to prevent, treat, or otherwise ameliorate astroke.

Bednar et al., Stroke 23(1):152 (1992) describe a rabbit model ofthromboembolic stroke wherein the arterial occlusion (an autologousblood clot delivered to the anterior cerebral circulation) is notremoved during the experiment. Rabbits received the binding protein(e.g., aLFA-1 binding antibody) or vehicle, 30 minutes following thethromboembolic event. Following embolization, the animals are evaluatedfor a total of 4 hours, including an initial 45 minutes of systemichypotension.

An aLFA-1 binding protein can be assayed for an effect on asthma oranother airway hyperresponsive disorder, e.g., using an animal modeldescribed in U.S. Pat. No. 5,730,983.

In one embodiment, a protein (e.g., an antibody described herein) has astatistically significant effect in an animal model. For example, theprotein has a statistically significant effect on a symptom of aninflammatory disorder, a disorder characterized by excessive LFA-1activity, or a LFA-1 mediated disorder.

Additional Assays

ELISA. Proteins encoded by a display library can also be screened for abinding property using an ELISA assay. For example, each protein iscontacted to a microtitre plate whose bottom surface has been coatedwith the target, e.g., a limiting amount of the target. The plate iswashed with buffer to remove non-specifically bound polypeptides. Thenthe amount of the protein bound to the plate is determined by probingthe plate with an antibody that can recognize the polypeptide, e.g., atag or constant portion of the polypeptide. The antibody is linked to anenzyme such as alkaline phosphatase, which produces a colorimetricproduct when appropriate substrates are provided. The protein can bepurified from cells or assayed in a display library format, e.g., as afusion to a filamentous bacteriophage coat. Alternatively, cells (e.g.,live or fixed) that express the target molecule, e.g., aconformationally biased LFA-1, can be plated in a microtitre plate andused to test the affinity of the peptides/antibodies present in thedisplay library or obtained by selection from the display library.

In another version of the ELISA assay, each polypeptide of a diversitystrand library is used to coat a different well of a microtitre plate.The ELISA then proceeds using a constant target molecule to query eachwell.

Homogeneous Binding Assays. The binding interaction of candidate proteinwith a target can be analyzed using a homogenous assay, i.e., after allcomponents of the assay are added, additional fluid manipulations arenot required. For example, fluorescence resonance energy transfer (FRET)can be used as a homogenous assay (see, for example, Lakowicz et al.,U.S. Pat. No. 5,631,169; Stavrianopoulos, et al., U.S. Pat. No.4,868,103). Another example of a homogenous assay is Alpha Screen(Packard Bioscience, Meriden Conn.).

Surface Plasmon Resonance (SPR). The binding interaction of a moleculeisolated from a display library and a target can be analyzed using SPR.SPR or Biomolecular Interaction Analysis (BIA) detects biospecificinteractions in real time, without labeling any of the interactants.Changes in the mass at the binding surface (indicative of a bindingevent) of the BIA chip result in alterations of the refractive index oflight near the surface (the optical phenomenon of surface plasmonresonance (SPR)). The changes in the refractivity generate a detectablesignal, which are measured as an indication of real-time reactionsbetween biological molecules. Methods for using SPR are described, forexample, in U.S. Pat. No. 5,641,640; Raether (1988) Surface PlasmonsSpringer Verlag; Sjolander and Urbaniczky (1991) Anal. Chem.63:2338-2345; Szabo et al. (1995) Curr. Opin. Struct. Biol. 5:699-705and on-line resources provide by BIAcore International AB (Uppsala,Sweden).

Information from SPR can be used to provide an accurate and quantitativemeasure of the equilibrium dissociation constant (K_(d)), and kineticparameters, including K_(on) and K_(off), for the binding of abiomolecule to a target. Such data can be used to compare differentbiomolecules. For example, proteins encoded by nucleic acid selectedfrom a library of diversity strands can be compared to identifyindividuals that have high affinity for the target or that have a slowK_(off). This information can also be used to develop structure-activityrelationships (SAR). For example, the kinetic and equilibrium bindingparameters of matured versions of a parent protein can be compared tothe parameters of the parent protein. Variant amino acids at givenpositions can be identified that correlate with particular bindingparameters, e.g., high affinity and slow K_(off). This information canbe combined with structural modeling (e.g., using homology modeling,energy minimization, or structure determination by crystallography orNMR). As a result, an understanding of the physical interaction betweenthe protein and its target can be formulated and used to guide otherdesign processes.

Protein Arrays. Polypeptides identified from the display library can beimmobilized on a solid support, for example, on a bead or an array. Fora protein array, each of the polypeptides is immobilized at a uniqueaddress on a support. Typically, the address is a two-dimensionaladdress. Protein arrays are described below (see, e.g., Diagnostics).

Cellular Assays. Candidate proteins can be selected from a library bytransforming the library into a host cell; the library could have beenpreviously identified from a display library. For example, the librarycan include vector nucleic acid sequences that include segments thatencode the polypeptides and that direct expression, e.g., such that thepolypeptides are produced within the cell, secreted from the cell, orattached to the cell surface. The cells can be screened or selected forpolypeptides that bind to the aLFA-1, e.g., as detected by a change in acellular phenotype or a cell-mediated activity. For example, in the caseof an antibody that binds to aLFA-1, the activity may be an in vitroassay for cell adhesion, cell invasion, or a lymphocyte activity.

Protein Production

Standard recombinant nucleic acid methods can be used to express anintegrin binding protein. See, for example, the techniques described inSambrook & Russell, Molecular Cloning: A Laboratory Manual, 3^(rd)Edition, Cold Spring Harbor Laboratory, N.Y. (2001) and Ausubel et al.,Current Protocols in Molecular Biology (Greene Publishing Associates andWiley Interscience, N.Y. (1989). Generally, a nucleic acid sequenceencoding the binding protein is cloned into a nucleic acid expressionvector. If the protein includes multiple polypeptide chains, each chaincan be cloned into an expression vector, e.g., the same or differentvectors, that are expressed in the same or different cells.

Antibody Production. Some antibodies, e.g., Fabs, can be produced inbacterial cells, e.g., E. coli cells. For example, if the Fab is encodedby sequences in a phage display vector that includes a suppressible stopcodon between the display entity and a bacteriophage protein (orfragment thereof), the vector nucleic acid can be transferred into abacterial cell that cannot suppress a stop codon. In this case, the Fabis not fused to the gene III protein and is secreted into the periplasmand/or media.

Antibodies can also be produced in eukaryotic cells. In one embodiment,the antibodies (e.g., scFv's) are expressed in a yeast cell such asPichia (see, e.g., Powers et al., 2001, J. Immunol. Methods.251:123-35), Hanseula, or Saccharomyces.

In one preferred embodiment, antibodies are produced in mammalian cells.Preferred mammalian host cells for expressing the clone antibodies orantigen-binding fragments thereof include Chinese Hamster Ovary (CHOcells) (including dhfr− CHO cells, described in Urlaub and Chasin, 1980,Proc. Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectablemarker, e.g., as described in Kaufman and Sharp, 1982, Mol. Biol.159:601 621), lymphocytic cell lines, e.g., NS0 myeloma cells and SP2cells, COS cells, and a cell from a transgenic animal, e.g., atransgenic mammal. For example, the cell is a mammary epithelial cell.

In addition to the nucleic acid sequence encoding the diversifiedimmunoglobulin domain, the recombinant expression vectors may carryadditional sequences, such as sequences that regulate replication of thevector in host cells (e.g., origins of replication) and selectablemarker genes. The selectable marker gene facilitates selection of hostcells into which the vector has been introduced (see e.g., U.S. Pat.Nos. 4,399,216, 4,634,665 and 5,179,017). For example, typically theselectable marker gene confers resistance to drugs, such as G418,hygromycin or methotrexate, on a host cell into which the vector hasbeen introduced. Preferred selectable marker genes include thedihydrofolate reductase (DHFR) gene (for use in dhfr⁻ host cells withmethotrexate selection/amplification) and the neo gene (for G418selection).

In an exemplary system for recombinant expression of an antibody, orantigen-binding portion thereof, a recombinant expression vectorencoding both the antibody heavy chain and the antibody light chain isintroduced into dhfr⁻ CHO cells by calcium phosphate-mediatedtransfection. Within the recombinant expression vector, the antibodyheavy and light chain genes are each operatively linked toenhancer/promoter regulatory elements (e.g., derived from SV40, CMV,adenovirus and the like, such as a CMV enhancer/AdMLP promoterregulatory element or an SV40 enhancer/AdMLP promoter regulatoryelement) to drive high levels of transcription of the genes. Therecombinant expression vector also carries a DHFR gene, which allows forselection of CHO cells that have been transfected with the vector usingmethotrexate selection/amplification. The selected transformant hostcells are cultured to allow for expression of the antibody heavy andlight chains and intact antibody is recovered from the culture medium.Standard molecular biology techniques are used to prepare therecombinant expression vector, transfect the host cells, select fortransformants, culture the host cells and recover the antibody from theculture medium. For example, some antibodies can be isolated by affinitychromatography with a Protein A or Protein G coupled matrix.

For antibodies that include an Fc domain, the antibody production systemmay produce antibodies in which the Fc region is glycosylated. Forexample, the Fc domain of IgG molecules is glycosylated at asparagine297 in the CH2 domain. This asparagine is the site for modification withbiantennary-type oligosaccharides. It has been demonstrated that thisglycosylation is required for effector functions mediated by Fcreceptors and complement Clq (Burton and Woof, 1992, Adv. Immunol.51:1-84; Jefferis et al., 1998, Immunol. Rev. 163:59-76). In oneembodiment, the Fc domain is produced in a mammalian expression systemthat appropriately glycosylates the residue corresponding to asparagine297. The Fc domain can also include other eukaryotic post-translationalmodifications.

Antibodies can also be produced by a transgenic animal. For example,U.S. Pat. No. 5,849,992 describes a method of expressing an antibody inthe mammary gland of a transgenic mammal. A transgene is constructedthat includes a milk-specific promoter and nucleic acids encoding theantibody of interest and a signal sequence for secretion. The milkproduced by females of such transgenic mammals includes,secreted-therein, the antibody of interest. The antibody can be purifiedfrom the milk, or for some applications, used directly.

One method for producing a transgenic mouse is as follows. Briefly, atargeting construct that encodes the antibody is microinjected into themale pronucleus of fertilized oocytes. The oocytes are injected into theuterus of a pseudopregnant foster mother for the development into viablepups. Some offspring incorporate the transgene.

It is also possible to produce antibodies that bind to aLFA-1 byimmunization, e.g., using an animal, e.g., with natural, human, orpartially human immunoglobulin loci. Non-human antibodies can also bemodified to include substitutions that insert human immunoglobulinsequences, e.g., consensus human amino acid residues at particularpositions, e.g., at one or more of the following positions (preferablyat least five, ten, twelve, or all): (in the FR of the variable domainof the light chain) 4L, 35L, 36L, 38L, 43L, 44L, 58L, 46L, 62L, 63L,64L, 65L, 66L, 67L, 68L, 69L, 70L, 71L, 73L, 85L, 87L, 98L, and/or (inthe FR of the variable domain of the heavy chain) 2H, 4H, 24H, 36H, 37H,39H, 43H, 45H, 49H, 58H, 60H, 67H, 68H, 69H, 70H, 73H, 74H, 75H, 78H,91H, 92H, 93H, and/or 103H (according to the Kabat numbering). See,e.g., U.S. Pat. No. 6,407,213.

Target Protein Production. Method for producing an conformationallybiased LFA-1 protein are described, e.g., in US 2002-0123614, Shimaoka,M et al. (2003) Cell 112,99-111; Shimaoka, M et al. (2002) Annu. Rev.Biophys. Biomol. Struct. 31, 485-516; and Shimaoka, M et al. (2001) ProcNatl Acad Sci USA 98:6009-6014 and Luo et al. (2003) Proc Natl Acad SciUSA. 100(5):2403-8.

Biotinylation Methods. A variety of methods are available to biotinylateproteins, e.g., an immunoglobulin protein or a target protein. Forexample, the protein can be incubated with a 5-fold molar excess ofsulfo-NHS-SS-biotin in 50 mM HEPES, pH 8.0, 100 mM NaCl overnight at 4°C. Free biotin is removed by buffer exchange into PBS, 0.01% Tween 20,e.g., using a BIOMAX™ device with a 10 kDa molecular weight cut-offmembrane or by dialysis. The number of biotin molecules incorporated permole of protein can be determined using the HABA assay as described bythe manufacturer (Pierce).

Pharmaceutical Compositions

In another aspect, the invention provides compositions, e.g.,pharmaceutically acceptable compositions, which include an integrinbinding protein, e.g., an antibody or other protein. The integrinbinding protein can be, e.g., a protein that preferentially binds toactivated LFA-1, formulated together with a pharmaceutically acceptablecarrier. As used herein, “pharmaceutical compositions” encompassdiagnostic compositions, e.g., labeled binding proteins (e.g., for invivo imaging) as well as therapeutic compositions.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like that arephysiologically compatible. The carrier can be suitable for intravenous,intramuscular, subcutaneous, parenteral, spinal or epidermaladministration (e.g., by injection or infusion). Depending on the routeof administration, the composition may be coated in a material toprotect the binding protein from the action of acids and other naturalconditions that may inactivate the binding protein.

A “pharmaceutically acceptable salt” refers to a salt that retains thedesired biological activity of the parent compound and does not impartany undesired toxicological effects (see e.g., Berge, et al. (1977) J.Pharm. Sci. 66:1-19). Examples of such salts include acid addition saltsand base addition salts. Acid addition salts include those derived fromnontoxic inorganic acids, such as hydrochloric, nitric, phosphoric,sulfuric, hydrobromic, hydroiodic, phosphorous and the like, as well asfrom nontoxic organic acids such as aliphatic mono- and dicarboxylicacids, phenyl-substituted alkanoic acids, hydroxy alkanoic acids,aromatic acids, aliphatic and aromatic sulfonic acids and the like. Baseaddition salts include those derived from alkaline earth metals, such assodium, potassium, magnesium, calcium and the like, as well as fromnontoxic organic amines, such as N,N′-dibenzylethylenediamine,N-methylglucamine, chloroprocaine, choline, diethanolamine,ethylenediamine, procaine and the like.

Compositions may be in a variety of forms. These include, for example,liquid, semi-solid and solid dosage forms, such as liquid solutions(e.g., injectable and infusible solutions), dispersions or suspensions,tablets, pills, powders, liposomes and suppositories. The form candepend on the intended mode of administration and therapeuticapplication. Typical compositions are in the form of injectable orinfusible solutions. One common mode of administration is parenteral(e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). Forexample, the integrin binding protein is administered by intravenousinfusion or injection. In another preferred embodiment, the integrinbinding protein is administered by intramuscular or subcutaneousinjection.

The phrases “parenteral administration” and “administered parenterally”as used herein mean modes of administration other than enteral andtopical administration. Parenteral administration is usually byinjection. Parenteral administration includes, e.g., intravenous,intramuscular, intraarterial, intrathecal, intracapsular, intraorbital,intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous,subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal,epidural and intrasternal injection and infusion.

Pharmaceutical compositions typically are sterile and stable under theconditions of manufacture and storage. A pharmaceutical composition canalso be tested to insure it meets regulatory and industry standards foradministration. For example, endotoxin levels in the preparation can betested using the Limulus amebocyte lysate assay (e.g., using the kitfrom Bio Whittaker lot # 7L3790, sensitivity 0.125 EU/mL) according tothe USP 24/NF 19 methods. Sterility of pharmaceutical compositions canbe determined using thioglycollate medium according to the USP 24/NF 19methods. For example, the preparation is used to inoculate thethioglycollate medium and incubated at 35° C. for 14 or more days. Themedium is inspected periodically to detect growth of a microorganism.

The composition can be formulated as a solution, microemulsion,dispersion, liposome, or other ordered structure suitable for deliveringa high concentration of the binding protein. Sterile injectablesolutions can be prepared by incorporating the binding protein in therequired amount in an appropriate solvent with one or a combination ofany other ingredients, followed by filtered sterilization. Generally,dispersions are prepared by incorporating the binding protein into asterile vehicle that contains a basic dispersion medium and any otheringredients. Sterile powders for the preparation of sterile injectablesolutions can be prepared by vacuum drying and freeze-drying. The properfluidity of a solution can be maintained, for example, by the use of acoating such as lecithin, by the maintenance of the required particlesize in the case of dispersion and by the use of surfactants. Prolongedabsorption of injectable compositions can be brought about by includingin the composition an agent that delays absorption, for example,monostearate salts and gelatin.

An integrin binding protein can be administered by any appropriatemethod. For many applications, the route of administration isintravenous injection or infusion. For example, for therapeuticapplications, the integrin binding protein can be administered byintravenous infusion. In certain embodiments, the binding protein may beprepared with a carrier that protects the protein against rapid release,such as a controlled release formulation, including implants, andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Manymethods for the preparation of such formulations are available. See,e.g., Sustained and Controlled Release Drug Delivery Systems, J. R.Robinson, ed., Marcel Dekker, Inc., New York, 1978.

To administer a compound described herein by other than parenteraladministration, it may be necessary to coat the compound with, orco-administer the compound with, a material to prevent its inactivation.The protein may be incorporated with excipients and used in the form ofingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. In certain embodiments, thebinding protein is administered orally, for example, with an inertdiluent or an assimilable edible carrier. The protein may also beenclosed in a hard or soft shell gelatin capsule, compressed intotablets, or incorporated directly into the subject's food or drink.

Pharmaceutical compositions can be administered by a medical device. Forexample, a pharmaceutical composition described herein can beadministered with a needleless hypodermic injection device, such as thedevices disclosed in U.S. Pat. Nos. 5,399,163, 5,383,851, 5,312,335,5,064,413, 4,941,880, 4,790,824, and 4,596,556. Examples of implants andmodules include: U.S. Pat. No. 4,487,603, which discloses an implantablemicro-infusion pump for dispensing medication at a controlled rate; U.S.Pat. No. 4,486,194, which discloses a therapeutic device foradministering medicants through the skin; U.S. Pat. No. 4,447,233, whichdiscloses a medication infusion pump for delivering medication at aprecise infusion rate; U.S. Pat. No. 4,447,224, which discloses avariable flow implantable infusion apparatus for continuous drugdelivery; U.S. Pat. No. 4,439,196, which discloses an osmotic drugdelivery system having multi-chamber compartments; and U.S. Pat. No.4,475,196, which discloses an osmotic drug delivery system.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. It is especially advantageousto formulate parenteral compositions in dosage unit form for ease ofadministration and uniformity of dosage. Dosage unit form as used hereinrefers to physically discrete units suited as unitary dosages for thesubjects to be treated; each unit contains a predetermined quantity ofbinding protein calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms can be dictated by and directly dependent on(a) the particular characteristics of the binding protein and theparticular therapeutic effect to be achieved, and (b) the sensitivity ofa particular individual.

An exemplary, non-limiting range for a therapeutically orprophylactically effective amount of an antibody described herein is0.01-20 mg/kg, e.g., 1-10, 0.01-10, 0.03-5, 0.02-2, or 0.01-1 mg/kg. Theintegrin binding protein, particularly an aLFA-1 binding antibody, canbe administered by intravenous infusion. It is to be noted that dosagevalues may vary with the type and severity of the condition to bealleviated. It is to be further understood that for any particularsubject, specific dosage regimens should be adjusted over time accordingto the individual need and the professional judgment of the personadministering or supervising the administration of the compositions, andthat dosage ranges set forth herein are exemplary only and are notintended to limit the scope or practice of the claimed composition.

A pharmaceutical composition may include a “therapeutically effectiveamount” or a “prophylactically effective amount” of an integrin bindingprotein, e.g., aLFA-1-binding protein. A “therapeutically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired therapeutic result. Atherapeutically effective amount of the composition may vary accordingto factors such as the disease state, age, sex, and weight of theindividual, and the ability of the protein to elicit a desired responsein the individual. A therapeutically effective amount is also one inwhich any toxic or detrimental effects of the composition are outweighedby the therapeutically beneficial effects. A “therapeutically effectivedosage” preferably inhibits a measurable parameter, e.g., parameter ofinflammation by at least about 5, 10, 20%, more preferably by at leastabout 40%, even more preferably by at least about 60%, and still morepreferably by at least about 80% relative to untreated subjects. Theability of a compound to inhibit a measurable parameter, e.g., aparameter of inflammation, can be evaluated in an animal model system ofinflammation. Alternatively, this property of a composition can beevaluated by examining the ability of the compound to inhibit, suchinhibition in vitro by assays known to the skilled practitioner.

A “prophylactically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredprophylactic result. Typically, since a prophylactic dose is used insubjects prior to or at an earlier stage of disease, theprophylactically effective amount will be less than the therapeuticallyeffective amount.

Stabilization and Retention

In one embodiment, an integrin binding protein (e.g., an aLFA-1-bindingantibody described herein or other integrin-binding protein) isphysically associated with a moiety that improves its stabilizationand/or retention in circulation, e.g., in blood, serum, lymph, or othertissues, e.g., by at least 1.5, 2, 5, 10, or 50 fold. For example, anaLFA-1 binding ligand can be associated with a polymer, e.g., asubstantially non-antigenic polymers, such as polyalkylene oxides orpolyethylene oxides. Suitable polymers will vary substantially byweight. Polymers having molecular number average weights ranging fromabout 200 to about 35,000 (or about 1,000 to about 15,000, and 2,000 toabout 12,500) can be used. For example, an aLFA-1 binding ligand can beconjugated to a water soluble polymer, e.g., hydrophilic polyvinylpolymers, e.g. polyvinylalcohol and polyvinylpyrrolidone. A non-limitinglist of such polymers include polyalkylene oxide homopolymers such aspolyethylene glycol (PEG) or polypropylene glycols, polyoxyethylenatedpolyols, copolymers thereof and block copolymers thereof, provided thatthe water solubility of the block copolymers is maintained. See, e.g.,U.S. Application Ser. No. 60/546,354, filed on Feb. 19, 2004, forfurther examples.

Treatments

Proteins that bind to an activated integrin, e.g., aLFA-1, havetherapeutic and prophylactic utilities. For example, these bindingproteins can be administered to a subject to treat or prevent adisorder, particularly inflammation, an inflammatory disorder, adisorder characterized by excessive LFA-1 activity, or a LFA-1 mediateddisorder.

A binding protein that preferentially binds to aLFA-1 can be used toprevent leukocytes with aLFA-1 from interacting with a cognate ligand ofLFA-1. The aLFA-1 binding protein can reduce the ability of theleukocyte from interacting with other cells or the extracellular matrix.For example, the binding protein can reduce the ability of the leukocyteto interact with an endothelial cell.

Because the binding protein preferentially binds to aLFA-1, a lowerconcentration of the binding protein may be effective to inhibiting suchinteractions, relative to the concentration required to achieve anequivalent effect using a binding protein that does not have apreference for aLFA-1 relative to non-activated conformations of LFA-1.

The integrin binding protein can be administered in an amount effectiveto ameliorate at least one symptom of inflammation, e.g., cause astatistically significant change in a parameter of inflammation.Exemplary parameters include: local temperature, core temperature,swelling (e.g., as measured), redness, local or systemic white bloodcell count, presence or absence of neutrophils, cytokine levels, andelastase activity. For quantitative parameters, the degree of change canbe, e.g., at least 10, 20, 30, 50, or 80%.

The integrin binding protein can be administered in an amount effectiveto reduce inflammation. Medical professionals can examine the subject toevaluate extent of inflammation.

The integrin binding protein can be administered in an amount effectiveto reduce leukocyte activity. Exemplary leukocyte activities includemigration and homing to sites on inflammation, adherence to theendothelium. In one embodiment, the binding protein is administeredlocally, e.g., to reduce local concentration of the leukocyte.

The integrin binding protein can be administered as part of a regimen,e.g., of multiple bolus doses. In one embodiment, the doses can alsoinclude the same (or within 20, or 10% of the same) amount of theprotein. In another embodiment, the initial dose is greater or less thanone or more subsequent doses, e.g., at least 10, 20, 50, 60, 70, or 80%greater or less than.

The dose can be selected or titrated, e.g., to achieve a detectableserum concentration whose mean trough concentration is less than 9, 7,6, 5, 4, 3, 2, 1, 0.3, 0.1, 0.03, or 0.01 μg/ml of the integrin bindingprotein.

As used herein, the term “treat” or “treatment” is the administration ofan integrin binding protein to a subject. The protein can beadministered alone or in combination with a second agent to a subject,an isolated tissue, or a cell. The protein can be administered toprevent or ameliorate the disorder, one or more symptoms of the disorderor a predisposition toward the disorder. Treating a cell includesmodulation of an activity (e.g., function or viability) of the cell.Exemplary functions of leukocytes that can be modulated include binding,migration, adhesion, and a T cell function. The modulation can reducethe ability of a cell to mediate a disorder, e.g., inflammation, aninflammatory disorder, a disorder characterized by excessive LFA-1activity, or a LFA-1 mediated disorder. Another example is an activitythat, directly or indirectly, reduces inflammation or an indicator ofinflammation. For example, the reduction can reduce a lymphocyteactivity.

An integrin binding protein can also be used to prevent a disorder,e.g., inflammation, inflammatory disorder, a disorder characterized byexcessive LFA-1 activity, or a LFA-1 mediated disorder. A prophylactictreatment can be effective, upon single- or multiple-dose administrationto the subject, in preventing or delaying the occurrence of the onset orrecurrence of a disorder, e.g., inflammation, an inflammatory disorder,a disorder characterized by excessive LFA-1 activity, or a LFA-1mediated disorder.

As used herein, the term “subject” includes human and non-human animals.Human subjects include a human patient having or suspected of having adisorder inflammation, inflammatory disorder, a disorder characterizedby excessive LFA-1 activity, or a LFA-1 mediated disorder.

The term “non-human animals” includes all vertebrates, e.g., non-mammals(such as chickens, amphibians, reptiles) and mammals, such as non-humanprimates, sheep, dog, cow, pig, etc. For example, the subject can be anon-human mammal that has cells that can express LFA-1 or an LFA-1-likeantigen with which an antibody described herein cross-reacts. Moreover,an aLFA-1-binding protein can be administered to a non-human mammalexpressing LFA-1 or an LFA-1-like antigen with which the binding proteininteracts(e.g., a primate, pig or mouse) for veterinary purposes or asan animal model of human disease. Regarding the latter, such animalmodels may be useful for evaluating the therapeutic efficacy of thebinding protein (e.g., testing of dosages and time courses ofadministration).

The aLFA-1-binding proteins can selectively inhibit, inactivate, or killcells that have activated LFA-1, e.g., to reduce inflammation, aleukocyte population, or leukocyte activity. For example, theaLFA-1binding protein can be conjugated to an agent, e.g., a cytotoxicagent such as a toxin, radioisotope, or short-range, high-energyα-emitters.

The aLFA-1 binding proteins can be used directly in vivo to inhibit,inactivate, or kill cells that present activated LFA-1 via naturalcomplement-dependent cytotoxicity (CDC) or antibody-dependent cellularcytotoxicity (ADCC). In one embodiment, the protein includes acomplement binding effector domain, such as an Fc portion (e.g.,functional portion) from IgG1, -2, or -3 or corresponding portions ofIgM which bind complement. Also encompassed by the invention is a methodof killing or ablating which involves using the aLFA-1 binding proteinsfor prophylaxis. For example, these materials can be used to prevent ordelay development or progression of inflammatory disease.

An aLFA-1 binding protein can be administered in combination with one ormore of the existing modalities for treating a disorder describedherein. “Combination” refers to the overlapping administration. Forexample, a subject may be receiving an aLFA-1 binding protein andanother therapy, e.g., another therapeutic agent, but the subject maynot be administered both therapies at the same instant. For example, thesubject may receive a first injection with the aLFA-1 binding protein,and then receive a separate injection with another therapeutic agent. Inanother example, the aLFA-1 binding protein and the other agent areadministered together in a single injection.

Regarding exemplary combinations, the aLFA-1 binding protein can used incombination with cyclosporins, rapamycins or ascomycins, or theirimmunosuppressive analogs, e.g. cyclosporin A, cyclosporin G, FK-506,rapamycin, 40-O-(2-hydroxy)ethyl-rapamycin etc.; corticosteroids;cyclophosphamide; azathioprene; methotrexate; brequinar; FTY 720;leflunomide; mnizoribine; mycophenolic acid; mycophenolate mofetil;15-deoxyspergualine; immunosuppressive monoclonal antibodies, e.g.,monoclonal antibodies to leukocyte receptors, e.g., MHC, CD2, CD3, CD4,CD7, CD25, CD28, B7, CD45, or CD58 or their ligands; or otherimmunomodulatory compounds, e.g. CTLA4Ig, or other adhesion moleculeinhibitors, e.g. mAbs or low molecular weight inhibitors includingselectin antagonists and VLA-4 antagonists. These combination therapiescan be part of an immunomodulating regimens or a regimen for thetreatment or prevention of allo- or xenograft acute or chronicrejection, an inflammatory disorder, or an autoimmune disorders.

In one embodiment, the aLFA-1 binding protein is administered to asubject to improve allo- or xenograft toleration. The protein can beadministered, before, during, and/or after the graft. The graft caninclude, e.g., skin, heart, liver, lung, or kidney tissue or organs. Forexample, the graft can include pancreatic cells. The aLFA-1 bindingprotein can be administered in combination with other agents, e.g.,agents that target CD154 or CD45RB, e.g., antibodies or solublereceptors that target these proteins, and/or rapamycin. See, e.g., Rayatet al. (2005) Diabetes 54:443-451.

Inflammatory Disorders

Exemplary inflammatory disorders include: acute and chronic immune andautoimmune pathologies (such as, but not limited to, rheumatoidarthritis (RA), juvenile chronic arthritis (JCA)), dermatologicaldiseases (such as psoriasis and contact dermatitis), graft versus hostdisease (GVHD), scleroderma, diabetes mellitus, allergy; asthma, acuteor chronic immune disease associated with an allogenic transplantation,such as, but not limited to, renal transplantation, cardiactransplantation, bone marrow transplantation, liver transplantation,pancreatic transplantation, small intestine transplantation, lungtransplantation and skin transplantation; chronic inflammatorypathologies such as, but not limited to, sarcoidosis, chronicinflammatory bowel disease, ulcerative colitis, and Crohn's pathology ordisease; multiple sclerosis; vascular inflammatory pathologies, such as,but not limited to, disseminated intravascular coagulation,atherosclerosis, Kawasaki's pathology and vasculitis syndromes, such as,but not limited to, polyarteritis nodosa, Wegener's granulomatosis,Henoch-Schonlein purpura, giant cell arthritis and microscopicvasculitis of the kidneys; chronic active hepatitis; Sjogren's syndrome;psoriatic arthritis; ophthalmic inflammatory diseases; enteropathicarthritis; reactive arthritis and arthritis associated with inflammatorybowel disease; infection diseases (such as septic shock, traumaticshock); and uveitis.

An aLFA-1-binding protein can be used to treat or prevent one of theforegoing diseases or disorders. For example, the protein can beadministered (locally or systemically) in an amount effective toameliorate at least one symptom of the respective disease or disorder.The protein may also ameliorate inflammation, e.g., a parameter ofinflammation, e.g., such as local temperature, swelling (e.g., asmeasured), redness, local or systemic white blood cell count, presenceor absence of neutrophils, cytokine levels, elastase activity, and soforth. It is possible to evaluate a subject, e.g., prior, during, orafter administration of the protein, for one or more of parameters ofinflammation, e.g., an aforementioned parameter.

IBD. Inflammatory bowel diseases (EBD) include generally chronic,relapsing intestinal inflammation. IBD refers to two distinct disorders,Crohn's disease and ulcerative colitis (UC). The clinical symptoms ofIBD include intermittent rectal bleeding, cramping abdominal pain,weight loss and diarrhea. A clinical index can also be used to monitorIBD such as the Clinical Activity Index for Ulcerative Colitis. Seealso, e.g., Walmsley et al. Gut. 1998 July; 43(1):29-32 and Jowett etal. (2003) Scand J Gastroenterol. 38(2):164-71. An integrin bindingprotein (e.g., an aLFA-1 binding antibody described herein) can be usedto ameliorate at least one symptom of IBD or to ameliorate a clinicalindex of IBD.

Psoriasis. Psoriasis is a chronic skin disease, characterized by scalingand inflammation. Psoriasis affects 1.5 to 2 percent of the UnitedStates population, or almost 5 million people. When psoriasis develops,typically patches of skin thicken, redden, and become covered withsilvery scales, referred to as plaques. Psoriasis most often occurs onthe elbows, knees, scalp, lower back, face, palms, and soles of thefeet. The disease also may affect the fingernails, toenails, and thesoft tissues inside the mouth and genitalia. About 10 percent of peoplewith psoriasis have joint inflammation that produces symptoms ofarthritis. The chronic skin inflammation of psoriasis is associated withhyperplastic epidermal keratinocytes and infiltrating mononuclear cells,including CD4+ memory T cells, neutrophils and macrophages.

Patients can be evaluated using a static Physician Global Assessment(sPGA), and receive a category score ranging from six categories betweenclear and very severe. The score is based on plaque, scaling, anderythema.

An integrin binding protein (e.g., an aLFA-1 binding antibody describedherein) can be used to ameliorate at least one symptom of psoriasis orto ameliorate a clinical index of psoriasis (e.g., sPGA index). Theprotein can be administered locally or systemically.

Rheumatoid Arthritis (RA). This disorder is characterized byinflammation in the lining of the joints and/or other internal organs.It is typically chronic, but can include flare-ups. Exemplary symptomsinclude inflammation of joints, swelling, difficulty moving, pain, lossof appetite, fever, loss of energy, anemia, lumps (rheumatoid nodules)under the skin, especially in areas subject to pressure (e.g., back ofelbows). In the clinical realm, rheumatoid arthritis (RA) is the mostcommon form of the severe arthrodysplastic disease. RA is a progressivedisease. An aLFA-1 binding protein can be used to ameliorate or preventat least one symptom of rheumatoid arthritis and other arthrodysplasticdisorders.

An aLFA-1 binding protein can be administered in conjunction withanother agent for treating rheumatoid arthritis, such as NSAIDs andaspirin, analgesics, and corticosteroids, help reduce joint pain,stiffness and swelling. Exemplary agents include disease-modifyinganti-rheumatic drugs (DMARDs) such as methotrexate, leflunomide,D-Penicillamine, sulfasalazine, gold therapy, minocycline, azathioprine,hydroxychloroquine (and other anti-malarials), cyclosporine, Prosorbatherapy, and biologic agents.

Asthma

Asthma is a heterogeneous family of diseases. It is characterized by ahyper-responsiveness of the tracheobronchi to stimuli (McFadden, E. R.et al., In: Harrison's Principles of Internal Medicine, 10th Ed.,Petersdorf, R. G. et al., Eds., McGraw-Hill, NY (1983), pages1512-1519); Kay, A. B., Allergy and Inflammation, Academic Press, NY(1987); which references are incorporated herein by reference).Clinically, asthma is manifested by the extensive narrowing of thetracheobronchi, by thick tenacious secretions, by paroxysms of dyspnea,cough, and wheezing. Although the relative contribution of each of theseconditions is unknown, the net result is an increase in airwayresistance, hyperinflation of the lungs and thorax, abnormaldistribution of ventilation and pulmonary blood flow. The disease ismanifested in episodic periods of acute symptoms interspersed betweensymptom-free periods. The acute episodes result in hypoxia, and can befatal. Approximately 3% of the general world population suffers from thedisease.

As used herein, “asthma” refers to either allergic or idiosyncraticasthma. Allergic asthma is usually associated with a heritable allergicdisease, such as rhinitis, urticaria, eczema, etc. The condition ischaracterized by positive wheal-and-flare reactions to intradermalinjections of airborne antigens (such as pollen, environmental oroccupational pollutants, etc.), and increased serum levels of IgE. Thedevelopment of allergic asthma appears to be causally related to thepresence of IgE antibodies in many patients. Asthma patients who do notexhibit the above-described characteristics are considered to haveidiosyncratic asthma.

An integrin binding protein (e.g., an aLFA-1 binding antibody describedherein) can be used to ameliorate at least one symptom of asthma or toameliorate a clinical index of asthma (e.g., airway responsiveness). Theprotein can be administered locally (e.g., by inhalation) orsystemically (e.g., by injection).

Ischemia/Stroke and Other Cardiovascular Disorders

The binding proteins described herein can also be used to treat orprevent cardiovascular disorders in which LFA-1 is a factor. Suchdisorders include, e.g., ischemia/reperfusion injury, e.g.,leukocyte-mediated reperfusion damage (e.g., post thrombolytic therapy),myocardial infarction, stroke, gut ischemia, and renal failure orhemorrhage shock.

An integrin binding protein (e.g., an aLFA-1 binding antibody describedherein) can be administered to a subject who is at risk for one of theabove disorders, e.g., at risk for a stroke, or to a subject who had astroke or other cardiovascular dysfunction. For example, the bindingprotein can be administered before, during, or immediately after, or anyother time after such a stroke or other cardiovascular dysfunction,e.g., within 2, 4, 6, 12, 24, or 48 hours. In one embodiment, thebinding protein is administered to reach a desired circulatingconcentration for at least 1, 2, 4, 5, 7, or 10 days.

An integrin binding protein (e.g., aLFA-1 binding protein, e.g., anaLFA-1 binding antibody described herein) can be used to treat a focalischemic stroke, e.g., a thromoboembolic stroke, or a cerebral ischemicstroke. “Focal ischemic stroke” is defined as damage to the brain causedby interruption of the blood supply to a region, generally caused byobstruction of any one or more of the “main cerebral arteries” (e.g.middle cerebral artery, anterior cerebral artery, posterior cerebralartery, internal carotid artery, vertebral artery or basilar artery).The “arterial obstruction” is generally a single embolus or thrombus. Acerebral embolism stroke can result from the obstruction of secondaryarteries or arterioles, e.g., as in the model of Bowes et al., Neurology45:815-819 (1995) in which a plurality of clot particles occludesecondary arteries or arterioles.

The aLFA-1 binding protein can be administered to increase cerebralblood flow can be increased and/or reduce infarct size in a subjecthaving suffered the stroke. The administering can be provided, e.g.,prior to removal of the arterial obstruction. For example, theobstruction is not removed until a therapeutic benefit, e.g., such asincreased cerebral blood flow is detected. The method can be performedwithout administering a thrombolytic agent.

The aLFA-1 binding protein can be administered to a patient as soon aspossible once the condition of acute ischemic stroke has been diagnosed,e.g., as suggested by focal deficit on neurologic examination.Neurologic examination and, optionally, neuro-imaging techniques such ascomputed tomography (CT) and magnetic resonance imaging (MRI) (includingdiffusion weighted imaging (DWI) and perfusion imaging (PI)); vascularimaging (e.g., duplex scanning and transcranial Doppler ultrasound andlaser Doppler); angiography (e.g., computerized digital subtractionangiography (DSA) and MR angiography) as well as other invasive ornon-invasive techniques can be used to diagnose acute ischemic stroke.

The aLFA-1 binding protein can be administered at least once orcontinuously at any time from immediately following to about 24 hoursafter the onset of stroke. In certain embodiments, the aLFA-1 bindingprotein is first administered to the patient at a time between about 15minutes (or 30 minutes or 45 minutes) to about 5 hours (or 12 hours or24 hours) from the onset of stroke. For example, the aLFA-1 bindingprotein may be first administered by bolus dosage as soon as stroke isdiagnosed, followed by a subsequent bolus dosage of the antagonist (e.g.5-24 hours after the initial bolus dosage). In another example theprotein is administered continuously.

Cancer

An integrin binding protein (e.g., an aLFA-1 binding antibody describedherein) can be used to treat a proliferative disorder of T-cells, e.g.,a T cell leukemia or lymphoma. In one embodiment, the disorder is acutepromyelocytic leukemia. Other exemplary disorders that can be treatedinclude myeloid disorders, such as acute promyeloid leukemia (APML),acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML)(reviewed in Vaickus, L. (1991) Crit Rev. in Oncol./Hemotol. 11:267-97).Lymphoid malignancies that may be treated include, e.g., acutelymphoblastic leukemia (ALL), which includes B-lineage ALL and T-lineageALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL),hairy cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM).Additional forms of malignant lymphomas include, e.g., non-Hodgkin'slymphoma and variants thereof, peripheral T-cell lymphomas, adult T-cellleukemia/lymphoma (ATL), cutaneous T-cell lymphoma (CTCL), largegranular lymphocytic leukemia (LGF) and Hodgkin's disease.

Diagnostic Uses

Binding proteins that bind to an activated integrin (e.g., aLFA-1), canalso be used for diagnostics in vitro and in vivo. In one aspect, theinvention provides a diagnostic method for detecting the presence of anaLFA-1 in vitro or in vivo (e.g., in vivo imaging in a subject).

In one embodiment, the integrin binding protein is used to evaluate asample in vitro(e.g., a biological sample). The method includes: (i)contacting a sample with aLFA-1-binding protein; and (ii) detectingformation of a complex between the aLFA-1-binding protein and thesample. The method can also include contacting a reference sample (e.g.,a control sample) with the binding protein, and determining the extentof formation of the complex between the binding protein and the sample,relative to the same for the reference sample. A change, e.g., astatistically significant change, in the formation of the complex in thesample or subject relative to the control sample or subject can beindicative of the presence of aLFA-1 in the sample. Samples can beobtained by surgical or non-surgical methods.

Another method includes: (i) administering the aLFA-1-binding protein toa subject; and (ii) detecting formation of a complex between theaLFA-1-binding protein, and the subject. The detecting can includedetermining location or time of formation of the complex. In oneembodiment, the subject has, is suspected of having, or is at risk for adisorder described herein, e.g., an inflammatory disorder, a disordercharacterized by excessive LFA-1 activity, or a LFA-1 mediated disorder.

The aLFA-1-binding protein can be directly or indirectly labeled with adetectable substance to facilitate detection of the bound or unboundantibody. Suitable detectable substances include various enzymes,prosthetic groups, fluorescent materials, luminescent materials andradioactive materials.

Complex formation between the aLFA-1-binding protein and aLFA-1 can bedetected by measuring or visualizing either the binding protein bound tothe aLFA-1 or unbound binding protein. Conventional detection assays canbe used, e.g., an enzyme-linked immunosorbent assays (ELISA), aradioimmunoassay (RIA) or tissue immunohistochemistry. Further tolabeling the aLFA-1-binding protein, the presence of aLFA-1 can beassayed in a sample by a competition immunoassay utilizing standardslabeled with a detectable substance and an unlabeled aLFA-1-bindingprotein. In one example of this assay, the biological sample, thelabeled standards and the aLFA-1 binding agent are combined and theamount of labeled standard bound to the unlabeled binding protein isdetermined. The amount of aLFA-1 in the sample is inversely proportionalto the amount of labeled standard bound to the aLFA-1 binding agent.

Fluorophore and chromophore labeled binding proteins can be prepared.Since antibodies and other proteins absorb light having wavelengths upto about 310 nm, the fluorescent moieties should be selected to havesubstantial absorption at wavelengths above 310 nm and preferably above400 nm. A variety of suitable fluorescers and chromophores are describedby Stryer (1968) Science, 162:526 and Brand, L. et al. (1972) AnnualReview of Biochemistry, 41:843-868. The binding proteins can be labeledwith fluorescent chromophore groups by conventional procedures such asthose disclosed in U.S. Pat. Nos. 3,940,475, 4,289,747, and 4,376,110.One group of fluorescers having a number of the desirable propertiesdescribed above is the xanthene dyes, which include the fluoresceins andrhodamines. Another group of fluorescent compounds are thenaphthylamines. Once labeled with a fluorophore or chromophore, thebinding protein can be used to detect the presence or localization ofthe aLFA-1 in a sample, e.g., using fluorescent microscopy (such asconfocal or deconvolution microscopy).

Histological Analysis. Immunohistochemistry can be performed using thebinding proteins described herein. For example, in the case of anantibody, the antibody can be synthesized with a label (such as apurification or epitope tag), or can be detectably labeled, e.g., byconjugating a label or label-binding group. For example, a chelator canbe attached to the antibody. The antibody is then contacted to ahistological preparation, e.g., a fixed section of tissue that is on amicroscope slide. After an incubation for binding, the preparation iswashed to remove unbound antibody. The preparation is then analyzed,e.g., using microscopy, to identify if the antibody bound to thepreparation.

The antibody (or other polypeptide or peptide) can be unlabeled at thetime of binding. After binding and washing, the antibody is labeled inorder to render it detectable.

Protein Arrays. The aLFA-1-binding protein can also be immobilized on aprotein array. The protein array can be used as a diagnostic tool, e.g.,to screen medical samples (such as isolated cells, blood, sera,biopsies, and the like). Of course, the protein array can also includeother binding proteins, e.g., that bind to aLFA-1 or to other targetmolecules, such as hyaluronic acid.

Methods of producing protein arrays are described, e.g., in De Wildt etal. (2000) Nat. Biotechnol. 18:989-994; Lueking et al. (1999) Anal.Biochem. 270:103-111; Ge (2000) Nucleic Acids Res. 28, e3, I-VII;MacBeath and Schreiber (2000) Science 289:1760-1763; WO 01/40803 and WO99/51773A1. Proteins for the array can be spotted at high speed, e.g.,using commercially available robotic apparati, e.g., from GeneticMicroSystems or BioRobotics. The array substrate can be, for example,nitrocellulose, plastic, glass, e.g., surface-modified glass. The arraycan also include a porous matrix, e.g., acrylamide, agarose, or anotherpolymer.

For example, the array can be an array of antibodies, e.g., as describedin De Wildt, supra. Cells that produce the binding proteins can be grownon a filter in an arrayed format. Protein production is induced, and theexpressed proteins are immobilized to the filter at the location of thecell.

A protein array can be contacted with a labeled target to determine theextent of binding of the target to each immobilized polypeptide from thediversity strand library. If the target is unlabeled, a sandwich methodcan be used, e.g., using a labeled probed, to detect binding of theunlabeled target.

Information about the extent of binding at each address of the array canbe stored as a profile, e.g., in a computer database. The protein arraycan be produced in replicates and used to compare binding profiles,e.g., of a target and a non-target. Thus, protein arrays can be used toidentify individual members of the diversity strand library that havedesired binding properties with respect to one or more molecules.

An aLFA-1-binding protein described herein can also be used to detectingbinding of an aLFA-1 to an insoluble support. For example, a sample canbe immobilized on array, and aLFA-1 can be detected on the array usingthe aLFA-1-binding protein.

FACS. (Fluorescent Activated Cell Sorting). The aLFA-1-binding proteincan be used to label cells, e.g., cells in a sample (e.g., a patientsample). For example, the protein can be used to detect activatedintegrins on cells (e.g., activated LFA-1). The binding protein istypically physically associated with (or attachable to) a fluorescentcompound. The cells can then be sorted using fluorescent activated cellsorted (e.g., using a sorter available from Becton DickinsonImmunocytometry Systems, San Jose Calif.; see also U.S. Pat. Nos.5,627,037; 5,030,002; and 5,137,809). As cells pass through the sorter,a laser beam excites the fluorescent compound while a detector countscells that pass through and determines whether a fluorescent compound isattached to the cell by detecting fluorescence. The amount of labelbound to each cell can be quantified and analyzed to characterize thesample.

The sorter can also deflect the cell and separate cells bound by thebinding protein from those cells not bound by the binding protein. Theseparated cells can be cultured and/or characterized.

In vivo Imaging. Integrin binding proteins can be used to detect thepresence of cells that include an activated integrin, e.g., cellspresenting aLFA-1, in vivo. The method includes (i) administering to asubject (e.g., a patient having an inflammatory disorder, a disordercharacterized by excessive LFA-1 activity, or a LFA-1 mediated disorder)an aLFA-1-binding antibody, conjugated to a detectable marker; (ii)exposing the subject to a means for detecting the detectable marker. Forexample, the subject is imaged, e.g., by NMR or other tomographic means.

Examples of labels useful for diagnostic imaging include radiolabelssuch as ¹³¹I, ¹¹¹In, ¹²³I, ^(99m)Tc, ³²P, ¹²⁵I, ³H, ¹⁴C, and ¹⁸⁸Rh,fluorescent labels such as fluorescein and rhodamine, nuclear magneticresonance active labels, positron emitting isotopes detectable by apositron emission tomography (“PET”) scanner, chemiluminescers such asluciferin, and enzymatic markers such as peroxidase or phosphatase.Short-range radiation emitters, such as isotopes detectable byshort-range detector probes can also be employed. The binding proteincan be labeled with such reagents using known techniques. For example,see Wensel and Meares (1983) Radioimmunoimaging and Radioimmunotherapy,Elsevier, New York for techniques relating to the radiolabeling ofantibodies and D. Colcher et al. (1986) Meth. Enzymol. 121: 802-816.

A radiolabeled binding protein can also be used for in vitro diagnostictests. The specific activity of a isotopically-labeled binding proteindepends upon the half-life, the isotopic purity of the radioactivelabel, and how the label is incorporated into the antibody.

Procedures for labeling polypeptides with the radioactive isotopes (suchas ¹⁴C, ³H, ³⁵S, ¹²⁵I, ³²P, ¹³¹I) are generally known. For example,tritium labeling procedures are described in U.S. Pat. No. 4,302,438.Iodinating, tritium labeling, and ³⁵S labeling procedures, e.g., asadapted for murine monoclonal antibodies, are described, e.g., byGoding, J. W. (Monoclonal antibodies: principles and practice:production and application of monoclonal antibodies in cell biology,biochemistry, and immunology 2nd ed. London; Orlando: Academic Press,1986. pp 124-126) and the references cited therein. Other procedures foriodinating polypeptides, such as antibodies, are described by Hunter andGreenwood (1962) Nature 144:945, David et al. (1974) Biochemistry13:1014-1021, and U.S. Pat. Nos. 3,867,517 and 4,376,110. Exemplaryradio-isotopes that are useful for imaging include ¹²³I, ¹³¹I, ¹¹¹In,and ^(99m)Tc. Procedures for iodinating antibodies are described byGreenwood, F. et al. (1963) Biochem. J. 89:114-123; Marchalonis, J.(1969) Biochem. J. 113:299-305; and Morrison, M. et al. (1971)Immunochemistry 289-297. Procedures for ^(99m)Tc-labeling are describedby Rhodes, B. et al. in Burchiel, S. et al. (eds.), Tumor Imaging: TheRadioimmunochemical Detection of Cancer, New York: Masson 111-123 (1982)and the references cited therein. Procedures suitable for ¹¹¹In-labelingantibodies are described by Hnatowich, D. J. et al. (1983) J. Immul.Methods, 65:147-157, Hnatowich, D. et al. (1984) J. Applied Radiation,35:554-557, and Buckley, R. G. et al. (1984) F.E.B.S. 166:202-204.

In the case of a radiolabeled binding protein, the binding protein isadministered to the patient, is localized to cells with which thebinding protein reacts, and is detected or “imaged” in vivo using knowntechniques such as radionuclear scanning using e.g., a gamma camera oremission tomography. See e.g., A. R. Bradwell et al., “Developments inAntibody Imaging”, Monoclonal Antibodies for Cancer Detection andTherapy, R. W. Baldwin et al., (eds.), pp 65-85 (Academic Press 1985).Alternatively, a positron emission transaxial tomography scanner, suchas designated Pet VI located at Brookhaven National Laboratory, can beused where the radiolabel emits positrons (e.g., ¹¹C, ¹⁸F, ¹⁵O, and¹³N).

MRI Contrast Agents. Magnetic Resonance Imaging (MRI) uses NMR tovisualize internal features of living subject, and is useful forprognosis, diagnosis, treatment, and surgery. MRI can be used withoutradioactive tracer compounds for obvious benefit. Some MRI techniquesare summarized in EP-A-0 502 814. Generally, the differences related torelaxation time constants T1 and T2 of water protons in differentenvironments are used to generate an image. However, these differencescan be insufficient to provide sharp high resolution images.

The differences in these relaxation time constants can be enhanced bycontrast agents. Examples of such contrast agents include a number ofmagnetic agents paramagnetic agents (which primarily alter T1) andferromagnetic or superparamagnetic (which primarily alter T2 response).Chelates (e.g., EDTA, DTPA and NTA chelates) can be used to attach (andreduce toxicity) of some paramagnetic substances (e.g., Fe⁺³, Mn⁺²,Gd⁺³). Other agents can be in the form of particles, e.g., less than 10μM to about 10 nM in diameter). Particles can have ferromagnetic,anti-ferromagnetic or superparamagnetic properties. Particles caninclude, e.g., magnetite (Fe₃O₄), □-Fe₂O₃, ferrites, and other magneticmineral compounds of transition elements. Magnetic particles mayinclude: one or more magnetic crystals with and without nonmagneticmaterial. The nonmagnetic material can include synthetic or naturalpolymers (such as sepharose, dextran, dextrin, starch and the like

The aLFA-1-binding proteins can also be labeled with an indicating groupcontaining of the NMR-active ¹⁹F atom, or a plurality of such atomsinasmuch as (i) substantially all of naturally abundant fluorine atomsare the ¹⁹F isotope and, thus, substantially all fluorine-containingcompounds are NMR-active; (ii) many chemically active polyfluorinatedcompounds such as trifluoracetic anhydride are commercially available atrelatively low cost, and (iii) many fluorinated compounds have beenfound medically acceptable for use in humans such as the perfluorinatedpolyethers utilized to carry oxygen as hemoglobin replacements. Afterpermitting such time for incubation, a whole body MRI is carried outusing an apparatus such as one of those described by Pykett (1982)Scientific American, 246:78-88 to locate and image activated leukocytes.

Information obtained from evaluating an aLFA-1-binding protein, e.g., abinding protein described herein, can be recorded on machine-compatiblemedia, e.g., computer readable or computer accessible media. Theinformation can be stored as a computer representation, e.g., in adatabase (e.g., in the case of imaging using a binding protein, adatabase of images for one or a plurality of subjects). The teen“computer representation” refers to information which is in a form thatcan be manipulated by a computer. The act of storing a computerrepresentation refers to the act of placing the information in a formsuitable for manipulation by a computer.

Kits

Also within the scope of the invention are kits that include acomposition described herein, e.g., a composition that contains anaLFA-1-binding protein. In one embodiment, the kit includes (a) acomposition that includes the aLFA-1-binding protein, and, optionally,(b) informational material. The informational material can bedescriptive, instructional, marketing or other material that relates tothe methods described herein and/or the use of the compound for themethods described herein, e.g., a treatment, prophylactic, or diagnosticuse. For example, the informational material describes methods foradministering the composition to treat a disorder, e.g., an inflammatorydisorder, a disorder characterized by excessive LFA-1 activity, or otherLFA-1 mediated disorder.

In one embodiment, the informational material can include instructionsto administer the compound in a suitable manner, e.g., in a suitabledose, dosage form, or mode of administration (e.g., a dose, dosage form,or mode of administration described herein). In another embodiment, theinformational material can include instructions for identifying asuitable subject, e.g., a human, e.g., a human having, or at risk for aninflammatory disorder, a disorder characterized by excessive LFA-1activity, or a LFA-1 mediated disorder. The informational material caninclude information about production of the compound, molecular weightof the compound, concentration, date of expiration, batch or productionsite information, and so forth. The informational material of the kitsis not limited in its form. Information about the compound can includestructural information, e.g., amino acid sequence, tradename, FDAapproved name, antibody isotype, and so forth. In many cases, theinformational material, e.g., instructions, is provided in printedmatter, e.g., a printed text, drawing, and/or photograph, e.g., a labelor printed sheet. However, the informational material can also beprovided in other formats, such as computer readable material, videorecording, or audio recording. In another embodiment, the informationalmaterial of the kit is a link or contact information, e.g., a physicaladdress, email address, hyperlink, website, or telephone number, where auser of the kit can obtain substantive information about the compoundand/or its use in the methods described herein. The informationalmaterial can also be provided in any combination of formats.

In addition to the composition that includes the aLFA-1-binding protein,the composition itself can include other ingredients, such as a solventor buffer, a stabilizer or a preservative, and/or a second agent fortreating a condition or disorder described herein, e.g., an inflammatorydisorder, a disorder characterized by excessive LFA-1 activity, or aLFA-1 mediated disorder. Alternatively, such other ingredients can beincluded in the kit, but in different compositions or containers thanthe composition that includes the aLFA-1-binding protein. In suchembodiments, the kit can include instructions for admixing the compoundand the other ingredients, or for using the compound together with theother ingredients.

The composition that includes the aLFA-1-binding protein can be providedin any form, e.g., liquid, dried or lyophilized form. The compositioncan be substantially pure and/or sterile. When the composition thatincludes the aLFA-1-binding protein is provided in a liquid solution,the liquid solution preferably is an aqueous solution, with a sterileaqueous solution being preferred. When the composition that includes theaLFA-1-binding protein is provided as a dried form, reconstitutiongenerally is by the addition of a suitable solvent. The solvent, e.g.,sterile water or buffer, can optionally be provided in the kit.

The kit can include one or more containers for the composition thatincludes the aLFA-1-binding protein. In some embodiments, the kitcontains separate containers, dividers or compartments for thecomposition and informational material. For example, the composition canbe contained in a bottle, vial, or syringe, and the informationalmaterial can be contained in a plastic sleeve or packet. In otherembodiments, the separate elements of the kit are contained within asingle, undivided container. For example, the composition is containedin a bottle, vial or syringe that has attached thereto the informationalmaterial in the form of a label. In some embodiments, the kit includes aplurality (e.g., a pack) of individual containers, each containing oneor more unit dosage forms (e.g., a dosage form described herein) of theaLFA-1-binding protein. For example, the kit includes a plurality ofsyringes, ampules, foil packets, or blister packs, each containing asingle unit dose of the compound. The containers of the kits can be airtight, waterproof (e.g., impermeable to changes in moisture orevaporation), and/or light-tight.

Kits can be provided that include an aLFA-1-binding antibody andinstructions for diagnostic, e.g., the use of the aLFA-1-binding protein(e.g., antibody or antigen-binding fragment thereof, or otherpolypeptide or peptide) to detect aLFA-1, in vitro, e.g., in a sample,e.g., a biopsy or cells from a patient having a disorder describedherein, or in vivo, e.g., by imaging a subject. The kit can furthercontain a least one additional reagent, such as a label or additionaldiagnostic agent. For in vivo use the ligand can be formulated as apharmaceutical composition.

The following invention is further illustrated by the followingexamples, which should not be construed as limiting.

EXAMPLES

The D2-57 Fab was isolated by depleting a Fab phage display library onthe low affinity wild type purified I-domain protein followed bypositive selection on high affinity locked open form I-domain LFA-1protein. D2-57 Fab binds to the high affinity locked open state ofpurified I-domain protein in the presence, but not absence of magnesium.It does not bind significantly to the low affinity locked closed stateof purified I-domain protein in either the presence or absence ofmagnesium.

In the presence of magnesium, D2-57 Fab binds to cells expressing wholeLFA-1 in which the α subunit contains an I-domain in the high affinitylocked open state (“HA cells”), but not when magnesium is absent. D2-57also binds in the presence of magnesium to activated wild-type LFA-1protein expressed on cells. D2-57 does not bind to cells expressingwhole LFA-1 when the α subunit contains an I-domain in the low affinitylocked closed state (“LA cells”). In contrast, MHM24 binds to both HAand LA cells.

The reformatted full IgG1, when tested with cells, has the same bindingspecificities as the Fab form.

Phage that display the P1-G10 in a Fab format bind to the high affinitylocked open form I-domain LFA-1 protein in the presence or absence ofmagnesium, but do not bind to the low affinity locked closed state.

The C1-54 Fab binds to both the high affinity locked open form I-domainLFA-1 protein and the low affinity locked closed state, butpreferentially binds to the open form in the presence of magnesium.

The following is a comparison of the variable region of exemplaryantibodies:

                                VARIABLE REGION - LIGHT CHAINS                FR1-L                CDR1-L       FR2-L        CDR2-LD2-57 QDIQMTQSPSSLSASVGDRVTITC RASQSIGSYLN WYQQKTGKAPKALIY AASSLQS C1-54QDIQMTQSPATLSVSPGERVTLSC TASQSVDSNLA WYQQKPGQAPRLLVY GASTRAT P1-G10QSV.LTQ.PPSVSVSPGQTASVTC SGDALGQKYAS WYQQKPGQSPVLVIF QDSKRPS                   FR3-L                   CDR3-L     FR4-L D2-57GVPSRFSGSGSGTDFTLTISSLQLEDFATYYC QQSYSTP..S FGQGTKLEIKRT C1-54GVPARFSGSGSGTAFTLTIDSLQSEDFAVYYC QQYNKWPPYS FGQGTKLEIKRT P1-G10GIPERFSGSNSGNTATLTISGTQAVDEADYYC QAWDTT.AYV FGTGTKVTVLSEQ ID NO: 33, 34, and 35 respectively.                                VARIABLE REGION - HEAVY CHAINS                FR1-H                  CDR1-H    FR2-H D2-57EVQLLESGGGLVQPGGSLRLSCAASGFTFS RYVMW WVRQAPGKGLEWVS C1-54EVQLLESGGGLVQPGGSLRLSCAASGFTFS HYGMS WVRQAPGKGLEWVS P1-G10EVQLLESGGGLVQPGGSLRLSCAASGFTFS HYSMQ WVRQAPGKGLEWVS                CDR2-H D2-57 YIWPSGGNTYYADSVKG C1-54 VISPSGGRTLYADSVKGP1-G10 YIGSSGGNTYYADSVKG                   FR3-H                    CDR3-H       FR4-H D2-57RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAS SYDFWSNAFDI WGQGTMVTVSS C1-54RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAK HYSY...AMDV WGQGTTVTVSS P1-G10RFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR G.TYNTSPFDY WGQGTLVTVSSSEQ ID NO: 36, 37, and 38 respectively.                      VARIABLE REGION - LIGHT CHAINS (Nucleic Acid)                           FR1-L D2-57CAAGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCAC C1-54CAAGACATCCAGATGACCCAGTCTCCAGCCACCCTGTCTGTGTCTCCAGGGGAAAGAGTCAC P1-G10CAGAGCGTCTTGA......CTCAGCCACCCTCAGTGTCCGTCTCCCCAGGACAGACAGCCAG                         CDR1-L D2-57CATCACTTGC CGGGCAAGTCAGAGCATTGGCAGCTACTTAAAC TGGTATCAGCAGAAAAC C1-54CCTCTCCTGC ACGGCCAGTCAGAGTGTTGACAGCAACTTAGCC TGGTATCAGCAAAAACC P1-G10CGTCACTTGC TCTGGAGATGCATTGGGACAA1AATATGCTTCC TGGTATCAACAGAAGCC               FR2-L                         CDR2-L D2-57AGGGAAAGCCCCTAAGGCCCTGATCTAT GCTGCATCCAGTTTGCAAAGT GGGGTCCCATC C1-54TGGCCAGGCTCCCAGACTCCTCGTCTAT GGTGCATCCACTAGGGCCACT GGTGTCCCAGC P1-G10AGGCCAGTCCCCTGTACTGGTCATCTTT CAAGATTCCAAGCGGCCCTCA GGGATCCCTGA                         FR3-L D2-57AAGGTTCAGTGGCAGTGGGTCTGGGACAGATTTCACTCTCACCATCAGTAGTCTGCAACTTG C1-54CAGGTTCAGTGGCAGTGGGTCTGGGACAGCGTTCACTCTCACCATCGACAGCCTGCAGTCTG P1-G10GCGGTTCTCTGGCTCCAATTCTGGGAACACAGCCACTCTGACCATCAGCGGGACCCAGGCTG                                   CDR3-L D2-57AAGATTTTGCAACTTACTACTGT CAACAGAGTTACA......GTACCCCCTCG TTCGGCC C1-54AAGATTTTGCAGTTTATTACTGT CAGCAGTATAATAAGTGGCCTCCGTACTCC TTTGGCC P1-G10TGGATGAGGCCGACTATTATTGT CAGGCGTGGGACA...CTACAGCTTATGTC TTCGGAA             FR4-L D2-57 AAGGGACCAAGGTGGAAATCAAA C1-54AGGGGACCAAGCTGGAGATCAAG P1-G10  CTGGGACCAAGGTCACCGTCCTASEQ ID NO: 39, 40, and 41 respectively.                      VARIABLE REGION - HEAVY CHAINS (Nucleic Acid)                           FR1-H D2-57GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTT C1-54GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTT P1-G10GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTT                                CDR1-H D2-57TCTTGCGCTGCTTCCGGATTCACTTTCTCT CGTTACGTTATGTGG TGGGTTCGCCAAGCT C1-54TCTTGCGCTGCTTCCGGATTCACTTTCTCT CATTACGGTATGTCT TGGGTTCGCCAAGCT P1-G10TCTTGCGCTGCTTCCGGATTCACTTTCTCT CATTACTCTATGCAG TGGGTTCGCCAAGCT            FR2-H                         CDR2-H D2-57CCTGGTAAAGGTTTGGAGTGGGTTTCT TATATCTGGCCTTCTGGTGGCAATACTTATTAT C1-54CCTGGTAAAGGTTTGGAGTGGGTTTCT GTTATCTCTCCTTCTGGTGGCCGTACTCTTTAT P1-G10CCTGGTAAAGGTTTGGAGTGGGTTTCT TATATCGGTTCTTCTGGTGGCAATACTTATTAT                                FR3-H D2-57GCTGACTCCGTTAAAGGT CGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTAC C1-54GCTGACTCCGTTAAAGGT CGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTAC P1-G10GCTGACTCCGTTAAAGGT CGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTAC                 FR3-H (contd) D2-57TTGCAGATGAACAGCTTAAGGGCTGAGGACACTGCAGTCTACTATTGTGCGAG TAGCTAC C1-54TTGCAGATGAACAGCTTAAGGGCTGAGGACACTGCAGTCTACTATTGTGCGAA ....... P1-G10 TTGCAGATGAACAGCTTAAGGGCTGAGGACACTGCAGTCTACTATTGTGCGAG AGGGACC            CDR3-H                   FR4-H D2-57GATTTTTGGAGTAATGCTTTTGATATC TGGGGCCAAGGGACAATGGTCACCGTCTCAAGC C1-54..ACATTACTCCTACGCTATGGACGTC TGGGGCCAAGGGACCACGGTCACCGTCTCAAGC P1-G10...TATAACACCTCCCCCTTTGACTAC TGGGGCCAGGGAACCCTGGTCACCGTCTCAAGCSEQ ID NO: 42, 43, and 44 respectively.

Exemplary ELISA data obtained with some of the described antibodies isas follows:

TABLE 2 Exemplary ELISA data HA with HA w/o LA with LA w/o Isolatenumber Mg²⁺ Mg²⁺ Mg²⁺ Mg²⁺ Control #1 0.15 0.10 0.13 0.07 C1-54 1.020.55 1.17 0.49 D2-57 0.68 0.09 0.13 0.07 P1-G10 0.87 1.06 0.16 0.08Control #2 0.17 0.10 0.14 0.07 no phage 0.10 0.08 0.08 0.06 P1-G10, nocells 0.05 0.06 0.05 0.05 blank 0.05 0.05 0.05 0.05

HA indicates the high affinity open form. LA indicates the low affinityclosed form of LFA-1. Control #1 refers to a phage that binds to adifferent target. Control #2 is another protein that binds to adifferent target. Numbers are in arbitrary units.

Germlining D2-57

The D2-57 light chain was compared to a human germline sequence. D2-57variants that include one or more alterations that increase the numberof similarities to a germline sequence (e.g., VKI-O2::JK1) can be used.For example, an antibody can include a D2-57 light chain with one ormore of the following substitutions, e.g., one, two, three, four, five,or six of the following substitutions (or insertion), e.g., atpositions: G30S, L40P, A46L, L80P, W96ins, and S97T. In many cases it ispreferable that A46 is maintained as an alanine. For example, theantibody can include an insertion that provides W96.

The C1-54 light chain was compared with a human germline sequence, e.g.,VKIII-L2. An antibody can include a C1-54 light chain with one or moreof the following substitutions, e.g., between one and eleven, two andfive, or six and eleven of the following substitutions (or deletion),e.g., at positions: D1E, Q3V, V19A, T24R, D30S, V48I, V58I, A70E, D76S,K93N, and P95a{tilde over (□)}

The P1-G10 light chain was compared with a human germline sequence,e.g., VL2-1 aka VL3 11-7. An antibody can include a P1-G10 light chainwith one or more of the following substitutions, e.g., between one andtwelve, two and five, or six and twelve of the following substitutions(or insertion), e.g., at positions: Q1S, S2Y, V3E, V21I, A28K, Q31aD,S34C, F49Y, V81M, T93S, T94S, and A95a(ins).

A vg3-23 related heavy chain can include one or more of the followingexemplary sequences in the JH region:

(SEQ ID NO: 45) JH1 ---AEYFQHWGQGTLVTVSS (SEQ ID NO: 46) JH2---YWYFDLWGRGTLVTVSS (SEQ ID NO: 47) JH3 -----AFDIWGQGTMVTVSS(SEQ ID NO: 48) JH4 -----YFDYWGQGTLVTVSS FDYWGQGTLVTVSS (SEQ ID NO: 49)JH5 ----NWFDPWGQGTLVTVSS (SEQ ID NO: 50) JH6 YYYYYGMDVWGQGTTVTVSS

An antibody can include a D2-57 heavy chain with one or more of thefollowing substitutions, e.g., between one and nine, two and five, orsix and nine of the following substitutions, e.g., at positions: R31S,V33A, W35S, Y50A, W52S, P52aG, N56S, S94R, A99Y, 1102Y, and M108L.Accordingly, the heavy chain variable domain can have fewer than ten,seven, six, or five differences relative to the germline sequence V3-23.

An antibody can include a C1-54 heavy chain with one or more of thefollowing substitutions, e.g., between one and seven, e.g., one, two,three, four, five, six, or seven of the following substitutions, e.g.,at positions: H31S, G33A, V50A, P52aG, R56S, L58Y, and K94R.Accordingly, the heavy chain variable domain can have fewer than seven,six, or five differences relative to the germline sequence V3-23.

An antibody can include a P1-G10 heavy chain with one or more of thefollowing substitutions, e.g., between one and seven, e.g., one, two,three, four, five, six, or seven of the following substitutions, e.g.,at positions: H31S, S33A, Q35S, Y50A, G52S, S52aG, and N56S.Accordingly, the heavy chain variable domain can have fewer than ten,seven, six, or five differences relative to the germline sequence V3-23.

Affinity Maturation of HC CDR3 of D2-57

Variants of the antibody were made that include variations in the CDR3region of the heavy chain variable domain. From such variants, cloneswere selected that bound to the high affinity (HA) I-domain, e.g., usingthe following conditions: 7.5 min binding time, 20 nM HA I-domain, and16 h incubation with 1 μM D2-57 IgG1 antibody. Some exemplary variantsthat bound to the LFA-1 I-domain in the activated conformation have thefollowing sequences in the CDR3 region of the heavy chain, from residues96 to 120 of the DX-2001 sequence:

         <--CDR3--->          12345678901 E0596 CASSYDLWSNAFDKWGQGTMVTVSS 120 from SEQ ID NO: 53 A0496 CASSYDLWSYAFEIWGQGTMVTVSS 120 from SEQ ID NO: 55 C0996 CASSYDYWSNAFDSWGQGTMVTVSS 120 from SEQ ID NO: 51 F0796 CASSFDFWSNAFDMWGQGTMVTVSS 120 from SEQ ID NO: 52 B0496 CASSYDFWSNAYANWGQGTMVTVSS 120 from SEQ ID NO: 56 F0596 CANSYDFRSNAFAVWGQGTMVTVSS 120 from SEQ ID NO: 54 C0296 CANSFDFWSNAFELWGQGTMVTVSS 120 from  **.*:* * *: ***********SEQ ID NO: 57

More complete sequences of these heavy chain variable domains are asfollows:

The C09 variant includes:

(SEQ ID NO: 51) EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYVMWWVRQAPGKGLEWVSYIWPSGGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASSYDYWSNAFDSWGQGTMVTVSS.

The F07 variant includes:

(SEQ ID NO: 52) EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYVMWWVRQAPGKGLEWVSYIWPSGGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASSFDRWSNAFDMWGQGTMVTVSS.

The E05 variant includes:

(SEQ ID NO: 53) EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYVMWWVRQAPGKGLEWVSYIWPSGGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASSYDLWSNAFDKWGQGTMVTVSS.

The F05 variant includes:

(SEQ ID NO: 54) EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYVMWWVRQAPGKGLEWVSYIWPSGGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCANSYDFRSNAFAVWGQGTMVTVSS.

The A04 variant includes:

(SEQ ID NO: 55) EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYVMWWVRQAPGKGLEWVSYIWPSGGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASSYDLWSYAFEIWGQGTMVTVSS.

The B04 variant includes:

(SEQ ID NO: 56) EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYVMWWVRQAPGKGLEWVSYIWPSGGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASSYDFWSNAYANWGQGTMVTVSS.

The CO₂ variant includes:

(SEQ ID NO: 57) EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYVMWWVRQAPGKGLEWVSYIWPSGGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCANSFDFWSNAFELWGQGTMVTVSS.

Without being bound by theory, the aspartic acid at position 3 in CDR3may interact with an Mg²⁺ ion bound to I-domain. This aspartic acid wasconserved 75 of 80 different affinity matured Fabs.

ICAM-1 blocking assay using HA cells with D2-57 (DX-1998.2) and 84different sFab isolates were performed. HA and LA cells were cultured inRPMI supplemented with 10% FCS, 100 U/ml penicillin-streptomycin, 2 mML-glutamine and 0.1 mM MEM non-essential amino acid at 37° C., 5% CO₂.Cells were harvested, washed once with HBS containing 10 mM EDTAfollowed by two washings with HBS. Cells were resuspended in activating(HBS, 10 mM MgCl₂, 2 mM EGTA) or inactivating buffer (HBS, 2 mM MgCl₂, 2mM CaCl₂) as indicated in the protocol. Myeloma IgG was added to blockthe Fc receptors. ICAM-1/SV-PE complex at a 4:1 ratio was added afterthe antibody addition. Cells were washed and resuspended in the stainingsolution after 30 mins. FACS analysis was done using the GUAVA EXPRESS™protocol.

In one assay using soluble Fabs, the IC₅₀s for three of the affinitymatured antibodies were 8.6±3.1 nM, 7.6±2.8 nM, and 6.5±2.6 nM, relativeto 11.5±4.8 nM for D2-57 in soluble Fab form as determined in parallel.

DX-2001

The following is an exemplary nucleic acid sequence that encodes thelight chains of DX-2001-light chain (variable and constant):

(SEQ ID NO: 58) GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTGGCAGCTACTTAAACTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGGCCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGGTCTGGGACAGATTTCACTCTCACCATCAGTAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTACAGTACCCCCTCGTTCGGCCAAGGGACCAAGGTGGAAATCAAAAGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGT.

The following is an exemplary nucleic acid sequence that encodes thelight chains of DX-2001-heavy chain (variable and constant):

(SEQ ID NO: 59) GAAGTTCAATTGTTAGAGTCTGGTGGCGGTCTTGTTCAGCCTGGTGGTTCTTTACGTCTTTCTTGCGCTGCTTCCGGATTCACTTTCTCTCGTTACGTTATGTGGTGGGTTCGCCAAGCTCCTGGTAAAGGTTTGGAGTGGGTTTCTTATATCTGGCCTTCTGGTGGCAATACTTATTATGCTGACTCCGTTAAAGGTCGCTTCACTATCTCTAGAGACAACTCTAAGAATACTCTCTACTTGCAGATGAACAGCTTAAGGGCTGAGGACACTGCAGTCTACTATTGTGCGAGTAGCTACGATTTTTGGAGTAATGCTTTTGATATCTGGGGCCAAGGGACAATGGTCACCGTCTCAAGCGCCTCCACCAAGGGCCCATCGGTCTTCCCGCTAGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTCCACACCTTCCCGGCTGTCCTACAGTCCTCCGGACTCTACTCCCTCAGCAGCGTAGTGACCGTGCCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATGCCCATCATGCCCAGCACCTGAGTTCCTGGGGGGACCATCAGTCTTCCTGTTCCCCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCCTCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGGTGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTGGCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGTAAATGA.

The following is an exemplary amino acid sequence of a DX-2001-lightchain (variable and constant):

(SEQ ID NO: 60) DIQMTQSPSSLSASVGDRVTITCRASQSIGSYLNWYQQKPGKAPKALIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTPSFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC.

The following is an exemplary amino acid sequence of a DX-2001-heavychain (variable and constant) IgG4:

(SEQ ID NO: 61) EVQLLESGGGLVQPGGSLRLSCAASGFTFSRYVMWWVRQAPGKGLEWVSYIWPSGGNTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASSYDFWSNAFDIWGQGTMVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK.

The changes in the light chain include altering position 40 from Thr toPro in framework 2 and/or altering position 80 from Leu to Pro inframework 3. Preferably, position 46 in framework 2 is Ala (rather thanthe germline Leu).

DX2001 (a germlined variant of D2-57, IgG4) binds to a small percentageof PBMCs (peripheral blood mononuclear cells) that are in MgCl₂/EGTAconditions. The binding is drastically enhanced by 20-min treatment withPMA (10 ng/ml) or DTT (500 μM). Treatment with PMA or DTT induces highaffinity state LFA-1. ICAM-1 has similar binding properties to PBMCs.

The PKC inhibitors, GF109203X and staurosporine, blocked PMA-inducedbinding of DX-2001 to PBMCs.

Species Specificity

Whole blood from healthy animals (rat, sheep, rabbit, dog, rhesusmonkey, cynomologus monkey and chimpanzee) were obtained from ValleyBiomedical Products and Services, Inc. PBMCs were isolated from wholeblood and cultured in low adherent 6-well plates overnight in RPMI 1640with 10%113S, 1× Pen/Strep. PBMCs were harvested, washed once withHanks' Balanced Salt Solution (HBSS) 1×, 20 mM HEPES buffer containing10 mM EDTA, and then twice with HBS. Cells were resuspended in HBSS at2×10⁶ cells/ml, 100 μl aliquots per well were added into 96-well plates(Costar round bottom). After spinning down, cell pellets wereresuspended in 50 μl activating buffer (HBSS, 10 mM MgCl₂, 2 mM EGTA) orinactivating buffer (HESS, 2 mM CaCl₂, 2 mM MgCl₂). For certain wells,PMA or DTT was added to a final concentration of 10 ng/ml or 500 μM,respectively. Cells were then incubated at 37° C. for 20 minutes. Cellswere incubated with DX-2001 (10 μg/ml) or a negative control antibodyFc-A2 (anti-CD44-Fc IgG₄ 10 μg/ml).

Antibodies that recognized CD-11a from the particular species (human,sheep, dog, rat, rabbit) (1:100 dilution) were used as positivecontrols. After incubation at room temperature for 30 minutes withgentle rocking, cells were washed twice with 220 μl/well HBSS containing0.05% NaN₃, and stained with PE-labeled secondary antibody at 100μl/well (anti-Human-IgG-PE, code: 709-116-149, Jackson Immunoresearch,and anti-mouse-IgG-PE, code: 115-115-164. Jackson Immunoresearch), 1:200diluted in HBSS. Cells were incubated at 4° C. for 30 minutes, thenwashed twice with 220 μl/well HBSS containing 0.05% NaN₃, resuspended in200 μl/well HBSS and analyzed using the GUAVA EXPRESS™ protocol (GuavaTechnologies, Inc., Hayward Calif.).

DX-2001 demonstrated good binding to chimpanzee PBMCs, at same extent asin human PBMCs. Binding to cells of these species is drasticallyincreased after PMA treatment. DX-2001 demonstrated minimal binding tocynomologus monkey, rat and sheep PBMCs. The binding in activatingconditions was increased slightly after PMA treatment. However, thebinding level is much less than in human and chimpanzee PBMCs. DX-2001does not show significant binding to rhesus monkey, dog and rabbitPBMCs. (summarized in Table 3).

TABLE 3 Species Cells No Treatment PMA DTT Human PBMCs ++ ++++ +++++++Chimpanzee PBMCs ++ ++++ ++ Cyn. monkey PBMCs + ++~+++ ++~+++ Rhesusmonkey PBMCs − − ND Dog PBMCs − − ND Rabbit PBMCs − − ND Rat PBMCs + ++ND Sheep PBMCs + ++ ND Mouse EL4 Cell line − ND ND The binding intensityof DX-2001 in hPBMCs without PMA was defined as “++”; after PMAtreatment defined as “++++”; DTT treatment defined as “++++++”. “−”: nobinding. N.D.: not determined. The bindings of all PBMCs from sevenspecies were compared with hPBMCs binding.

The ability of D2-57 IgG to inhibit I-CAM binding to HA cells (i.e.,cells expressing LFA-1 locked in the high affinity form) was evaluatedand compared to MHM24 IgG in parallel. IC₅₀ values were determined usingGUAVA™ analysis. In one experiment, the IC₅₀ values for D2-57 and MGM24IgG were 0.18±0.02 and 0.24±0.04 nM, respectively. In anotherexperiment, the IC₅₀ values for D2-57 and MGM24 IgG were 0.59±0.1 nM(first lot of D2-57 IgG) and 0.62±0.16 nM (second lot of D2-57 IgG) and1.8±0.8 nM (MGM24) respectively. In still another experiment, the IC₅₀values for D2-57 and MGM24 IgG were 0.24±0.02 and 0.59±0.13 nM,respectively. In still another experiment, the IC₅₀ values for D2-57 andMGM24 IgG were 0.68±0.2 and 2.4±0.8 nM, respectively. The pattern thatemerges from these experiments is that, within the margin ofexperimental error, D2-57 has an IC₅₀ that is as good or better thanMHM24 in this particular assay set-up.

The ability D2-57 IgG to inhibit I-CAM binding to human PBMCs was alsoevaluated. The average of four experiments indicated that the IC₅₀values for D2-57 and MGM24 IgG were 0.54±0.44 and 0.33±0.17 nM,respectively.

1. A method of inhibiting the binding of αLFA-1 to the ligand ICAM, themethod comprising: providing an αLFA-1 binding protein comprising anisolated antibody comprising an immunoglobulin heavy chain (HC) variabledomain and an immunoglobulin light chain (LC) variable domain, whereinthe HC variable domain and the LC variable domain form an antigenbinding site that binds to an activated conformation of LFA-1, whereinthe antibody comprises: (i) a heavy chain variable domain thatcomprises:(a) a CDR1 that comprises at RYVMW (SEQ ID NO: 1); (b) a CDR2that comprises YIWPSGGNTYYADSVKG (SEQ ID NO:2); and (c) a CDR3 thatcomprises a sequence selected from the group consisting of SEQ ID NO:3;residues 96-120 of SEQ ID NO:51; residues 96-120 of SEQ ID NO:52;residues 96-120 of SEQ ID NO:53; residues 96-120 of SEQ ID NO:54;residues 96-120 of SEQ ID NO:55; residues 96-120 of SEQ ID NO:56;residues 96-120 of SEQ ID NO:57; and a light chain variable domain thatcomprises (d) a CDR1 that comprises RASQSIGSYLN (SEQ ID NO:7); (e) aCDR2 that comprises AASSLQS (SEQ ID NO:8); and (f) a CDR3 that comprisesat QQSYSTPS (SEQ ID NO: 9); or (ii) a heavy chain variable domain thatthat comprises SEQ ID NO:23, SEQ ID NO:25; SEQ ID NO:27, or SEQ IDNO:20; and a light chain variable domain that comprises SEQ ID NO:22,SEQ ID NO:24, SEQ ID NO:26, or SEQ ID NO:28; or (iii) a heavy chainvariable domain that comprises a sequence encoded by a nucleic acid thathybridizes under high stringent conditions to the complement of the fulllength of SEQ ID NO:42, SEQ ID NO:43, or SEQ ID NO:44; and a light chainvariable domain that comprises a sequence encoded by a nucleic acid thathybridizes under high stringent conditions to the complement of the fulllength of SEQ ID NO:39, SEQ ID NO:40, or SEQ ID NO:41; or (iv) anantibody selected from the group consisting of a) an immunoglobulinheavy chain variable domain sequence comprising SEQ ID NO:23, and animmunoglobulin light chain variable domain sequence comprising SEQ IDNO:22; b) an immunoglobulin heavy chain variable domain sequencecomprising SEQ ID NO:25, and an immunoglobulin light chain variabledomain sequence comprising SEQ ID NO:24; c) an immunoglobulin heavychain variable domain sequence comprising SEQ ID NO:27, and animmunoglobulin light chain variable domain sequence comprising SEQ IDNO:26; and d) an immunoglobulin heavy chain variable domain sequencecomprising SEQ ID NO:29, and an immunoglobulin light chain variabledomain sequence comprising SEQ ID NO:28; and contacting the protein toαLFA-1 , in an amount sufficient to inhibit αLFA-1 binding to ICAM. 2.The method of claim 1 wherein the contacting is in vitro.
 3. The methodof claim 1 wherein the contacting is in vivo.
 4. The method of claim 1wherein the protein is contacted to αLFA-1 in the vicinity of aneoplastic cell.
 5. The method of claim 1 wherein the protein iscontacted to αLFA-1 in the vicinity of an endothelial cell.